Chronic lymphocytic leukemia (CLL) is a frequent lymphoproliferative disorder of B cells. Although inhibitors targeting signal proteins involved in B-cell antigen receptor (BCR) signaling constitute an important part of the current therapeutic protocols for CLL patients, the exact role of BCR signaling, as compared to genetic aberration, in the development and progression of CLL is controversial. In order to investigate whether BCR expression per se is pivotal for the development and maintenance of CLL B cells, we used the TCL1 mouse model. By ablating the BCR in CLL cells from TCL1 transgenic mice, we show that CLL cells cannot survive without BCR signaling and are lost within 8 weeks in diseased mice. Furthermore, we tested whether mutations augmenting B-cell signaling influence the course of CLL development and its severity. The phosphatidylinositol-3-kinase (PI3K) signaling pathway is an integral part of the BCR signaling machinery and its activity is indispensable for B-cell survival. It is negatively regulated by the lipid phosphatase PTEN, whose loss mimics PI3K pathway activation. Herein, we show that PTEN has a key regulatory function in the development of CLL, as deletion of the Pten gene resulted in greatly accelerated onset of the disease. By contrast, deletion of the gene TP53, which encodes the tumor suppressor p53 and is highly mutated in CLL, did not accelerate disease development, confirming that development of CLL was specifically triggered by augmented PI3K activity through loss of PTEN and suggesting that CLL driver consequences most likely affect BCR signaling. Moreover, we could show that in human CLL patient samples, 64% and 81% of CLL patients with a mutated and unmutated IgH VH, respectively, show downregulated PTEN protein expression in CLL B cells if compared to healthy donor B cells. Importantly, we found that B cells derived from CLL patients had higher expression levels of the miRNA-21 and miRNA-29, which suppresses PTEN translation, compared to healthy donors. The high levels of miRNA-29 might be induced by increased PAX5 expression of the B-CLL cells. We hypothesize that downregulation of PTEN by increased expression levels of miR-21, PAX5 and miR-29 could be a novel mechanism of CLL tumorigenesis that is not established yet. Together, our study demonstrates the pivotal role for BCR signaling in CLL development and deepens our understanding of the molecular mechanisms underlying the genesis of CLL and for the development of new treatment strategies.
Chronic lymphocytic leukemia (CLL) is the most frequent type of leukemia in Western countries.1 Like most neoplastic B-cell malignancies, CLL cells maintain their B-cell antigen receptor (BCR) expression.2 This selective pressure to maintain a functional BCR is linked to the fact that malignant B cells profit from the proliferation and survival signals triggered by the BCR.3 Several lines of evidence support a key role of BCR signaling in the pathogenesis of CLL. Thus, CLL with hyper-mutated immunoglobulin heavy chain variable region (IgH VH) genes (mutated [M]-CLL) show a more favorable prognosis than those with unmutated IgH VH genes (unmutated [U]-CLL).4 Indeed, patients with fewer than 2% mutations in the IgH VH genes present a more polyreactive BCR and have a more aggressive disease with shorter survival.5 Furthermore, the IgH VH repertoire is highly restricted leading to different groups of CLL patients with stereotypic BCR.6 In contrast to diffuse large B-cell lymphoma (DLBCL), the BCR-activating signaling is not due to mutations in BCR signaling components7 but to autonomously activated BCR signaling initiated by the ability of CLL BCR to bind each other.8 More recently the crystal structures of a set of CLL BCR identified the regions involved in this binding. Small molecule inhibitors against BTK, like ibrutinib and acalabrutinib show anti-tumor activity in clinical studies of relapsed/refractory CLL.9 In line with this, the inhibition of BTK kinase activity through targeted genetic inactivation and inhibition of BTK by ibrutinib in Eµ-TCL1 mouse models significantly delays the outbreak of CLL, demonstrating that BCR signaling is critical for CLL development and expansion.10
The remarkable clinical effectiveness of BCR signaling inhibitors underscores the importance of BCR signaling and of BCR-associated kinases in the proliferation and homing of CLL cells, making this class of agents the treatment of choice for CLL patients.11
The prolonged survival of CLL cells is in part associated with defective apoptosis triggered by the phosphatidylinositol 3-kinase (PI3K)/ protein kinase B (PKB/AKT) and NF-κB pathways, which, among other pathways, are downstream of the BCR.12 PI3K exerts its effects by generating phosphatidylinositol-3,4,5-trisphosphate (PI(3,4,5)P3) via phosphorylating the 3-position of the inositol ring of phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2).13 On the contrary, the phosphatase and tensin homolog deleted on chromosome 10 (PTEN) can dephosphorylate PI(3,4,5)P3 at the 3-position of the inositol ring converting PI(3,4,5)P3 back to PI(4,5)P2.13,14 Loss of PTEN results in accumulation of PI(3,4,5)P3 mimicking the effect of PI3K activation and triggering the activation of its downstream effector AKT.15 TCL1 is an oncoprotein contributing to occurrence of T-cell prolymphocytic leukemia, as a result of chromosomal translocations and inversions at 14q31.2.16 Although such a defect is not found in CLL, TCL1 is expressed in more than 90% human CLL patients.17
In order to facilitate the development of novel therapeutics for B-cell malignancies, an in vivo model, which recapitulates the human disease, is required. Several mouse models have provided important insights into CLL pathogenesis.18 These particularly include the widely studied Eµ-TCL1 model.19 Aging transgenic mice that overexpress TCL1 under the control of the µ immunoglobulin (Igµ) gene enhancer, develop a CD5-positive B-cell lymphoproliferative disorder mimicking human CLL and implicating TCL1 in the pathogenesis of CLL.20 Given the importance of intrinsic BCR signaling in survival and progression of CLL, the establishment of a mouse model that can provide a genetic answer to the importance of the BCR in CLL would be a benefit for the understanding of its pathogenesis.
In this study, we investigated the role of the BCR in the development of a CLL-like B-cell tumor disease in the mouse. The transformed B cells are here referred to as CLL cells. We also explored the impact of the PI3K signaling on the progression of the disease and found that Ptendeletion accelerated the onset of leukemogenesis. Moreover, we revealed that PTEN is downregulated in human CLL patients, which might be caused by increased expression levels of the microRNA family miR-29 and the protein PAX5. Thus, by generating inducible mouse models allowing the inactivation of BCR components we established a tool for the investigation of CLL signal transduction and treatment modalities. Our present data identify the BCR as a uniquely important regulator of CLL viability, confirm that an increased PI3K-signaling pathway supports CLL development and maintenance and describe PTEN as a potential target for therapeutic intervention.
In order to delete the mb-1 gene, encoding the Igα protein in B cells, the previously described mouse strain IgαTMF (Igαfl/fl),21 was crossed with the mb1-CreERT2 strain (Cd79atm3(cre/ERT2)Reth), which expresses a tamoxifen-inducible form of the Cre recombinase under the control of the mb-1 promoter region.22
Mb1-CreERT2; Igαfl/fl mice were crossed to the Eµ-TCL1 mouse strain20 to generate the compound mouse mb1-CreERT2;Igαfl/fl; Eµ-TCL1. The mb1-CreERT2;Eµ-TCL1 served as a control.
The Ptenfl/fl mouse strain23 was crossed with the mb1-CreERT2;Eµ-TCL1. Mice heterozygous for Pten (Ptenfl/+) were generated by crossing Ptenfl/fl mice24 to the mb1-Cre mouse (Cd79atm1(cre)Reth/Ehobj) to generate the mb1-Cre;Ptenfl/+ mouse strain. Treatment of the mice with anti-IL7R antibody (Ab) and tamoxifen as well as transfer and propagation of CLL cells in Rag2-/-;γc-/- mice is described in the Online Supplementary Appendix.
All mice were bred on Black6 background. All animal studies were carried out in accordance with the German Animal Welfare Act, having been reviewed by the regional council and approved under the license (#1288 L1-L18).
Culture of splenic cells
The splenic cells of Tam-treated mb1-CreERT2;Ptenfl/fl;Eµ-TCL1 mice were cultured in complete medium (ISCOVE’s, 10% fetal calf serum, 50 mg/mL gentamycin, 50 mM 2-mercapto-ethanol) without any additional growth factors (like BAFF) and incubated at 37°C in the presence of 7.5% CO2. Only in the case of ex vivo inactivation of PTEN expression in purified primary splenic B2 cells from mb1-CreERT2;Ptenfl/fl;Eµ-TCL1 mice and mb1-CreERT2;Eµ-TCL1 controls, the cells were cultured in the presence of 50 ng/mL recombinant human BAFF and were treated with 1 µM Tam (4-OHT).
For flow cytometric analysis, cell suspensions were pre-treated with α-CD16/CD32 Fc Block (2,4G2; BD Bio-sciences). Dead cells were excluded by staining with Fixable Viability Dye eFluor 450 (eBioscience). Intracellular (IC) flow cytometry staining was performed using the ADG Fix&Perm Kit (Dianova). The detailed IC and extracellular staining procedure including the respective antibodies is provided in the Online Supplementary Appendix. Cells were acquired at a FACS Canto II flow cytometer (BD Bio-sciences). Analysis was performed using the FlowJo software (Tree Star).
Ca2+ influx measurement
0.5–1×106 cells preloaded with the calcium-sensitive fluorescent dye Indo-1 (Invitrogen) were analyzed by flow cytometry (LSR Fortessa, BD Biosciences) upon application of 10 µg/mL anti-IgM F(ab')2 fragments (Jackson ImmunoResearch).
Chronic lymphocytic leukemia patient and healthy donor sample analysis
CLL samples were obtained from the Department of Internal Medicine III, University Hospital Ulm. Peripheral blood mononuclear cells from healthy donors (HD) were obtained from the Institute for Clinical Transfusion Medicine and Immunogenetics at Ulm University Medical Center. All samples were obtained with informed consent and used in full compliance with institutional regulations (no. 456/19).
Primary CLL patient and HD lymphocytes were isolated from peripheral blood using Ficoll-Paque PLUS (GE Healthcare, 17-440-03). The human samples were MACSed for CD19-positive B cells before microRNA (miRNA) and total RNA isolation for real-time quantitative polymerase chain reaction (qRT-PCR) analysis. More details are provided in the Online Supplementary Appendix.
Unpaired two-tailed Student’s t-tests (with n between 3 and 5 mice per group) were carried out using Prism 9 software (GraphPad Software Inc) to determine the statistical differences between groups.
Efficient inducible deletion of the Igα-encoding mb-1 gene in B cells of mb1-CreERT2; Igαbfl/fl mice
In order to investigate the efficiency of mb1-CreERT2 in the deletion of Igα in mature B cells we crossed mb1-CreERT2 with Igαfl/fl mice, in which the exon 3 and 4 of the mb-1 gene are flanked by loxP sites (floxed). All splenic B cells isolated from the tamoxifen (Tam)-treated mb1-CreERT2;Igαfl/fl mice lacked both IgM and IgD expression while splenic B cells from the mb1-CreERT2 mice treated in the same manner still expressed the BCR (Figure 1A and B). Furthermore, 8 weeks after the start of the Tam-treatment and additional treatment with anti-IL-7R Ab, which block the influx of newly generated B cells from the bone marrow (BM), absolute splenic B-cell numbers from mb1-CreERT2;Igαfl/fl mice were significantly reduced (up to 40×) when compared to those from the spleens of the mb1-CreERT2 mice (Figure 1C). Additionally, peripheral blood (PBL), lymph nodes (LN) and peritoneal cavity (PC) of these mice contained only a few surviving B cells (Figure 1D). In line with other studies these results demonstrate that mature B cells absolutely require the expression of Igα and consequently of the BCR for their survival in the periphery.
Inactivation of Igα in mouse chronic lymphocytic leukemia reverts the disease phenotype
Although inhibitors against prominent signaling molecules downstream of the BCR, like ibrutinib and idelalisib, successfully eradicate CLL cells in patients, pointing to the quintessential role of the BCR in CLL cell survival, there is no genetic evidence for the involvement of the BCR in the maintenance of CLL cells. Therefore, we generated a mouse model, which enables us to address this question. In order to investigate the role of the BCR in mouse CLL we crossed the mb1-CreERT2; Igαfl/fl mice with the Eµ-TCL1 mouse strain, which is a well-accepted model for mouse CLL, generating mb1-CreERT2;Igαfl/fl;Eµ-TCL1 mice. Over-expression of TCL1 in B cells drives the development of CLL cells over time. The disease is first detected in 6-month-old mice and its incidence increases with age, reaching its maximum at 12 months. The main features of mouse CLL are the expression of CD5 (Figure 2A; right panel) and the deregulation of B220 and IgD expression. Before and after the beginning of Tam treatment, we assessed the development and survival of CLL cells in the blood of the transgenic mice by flow cytometry using the mouse CLL key markers CD19+ CD93- B220low CD5+ IgM+
IgD-. Fully diseased mb1-CreERT2;Igαfl/fl;Eµ-TCL1 and control mb1-CreERT2; Eµ-TCL1 (12-month-old) carrying mainly CLL cells (Figure 2A) were sacrificed 8 weeks after the beginning of the Tam treatment and without additional treatment with anti-IL-7R Ab. The frequencies and absolute B-CLL cell numbers in the spleen differed dramatically and significantly between mb1-CreERT2;Igαfl/fl;Eµ-TCL1 and mb1-CreERT2;Eµ-TCL1 control mice (Figure 2A and C). The 100-fold decrease of cellularity after deletion of Igα resulted in a significant reduction of the spleen size and weight in mb1-CreERT2;Igαfl/fl;Eµ-TCL1 mice compared to the Igα-sufficient controls (Figure 2B). In the PBL and the BM (Online Supplementary Figure S1A to C), the frequencies and absolute cell numbers of mouse CLL cells differed significantly between mb1-CreERT2;Igαfl/fl;Eµ-TCL1 and mb1-CreERT2;Eµ-TCL1 control mice.
In order to further investigate the impact of Igα gene deletion on the overall survival of the diseased mice, immunodeficient Rag2-/-;γc-/- mice were intraperitoneally (i.p.) transplanted with splenic cells (1×107) from either mb1-CreERT2;Igαfl/fl;Eµ-TCL1 or mb1-CreERT2;Eµ-TCL1 mice, which were previously sequentially transferred in Rag2-/-;γc-/-. The mice were treated with Tam three-times every third day, without additional treatment of anti-IL-7R Ab, and were analyzed at day 15 after the start of the treatment. This model has the advantage of producing a CLL-like phenotype including peripheral blood leukemia and splenomegaly in a short period of time, compared to the long period in the original TCL1 model. While Rag2-/-;γc-/- mice transplanted with mb1-CreERT2;Eµ-TCL1 CLL cells died already between day 12 and 18, the mice transplanted with the mb1-CreERT2;Igαfl/fl; Eµ-TCL1 CLL cells survived up to day 30 (Figure 2D).
Notably, as shown in the previous experiments, BCR-deficient CLL cells of the mb1-CreERT2;Igαfl/fl;Eµ-TCL1 genotype were significantly reduced in the spleens of the Tam-treated Rag2-/-;γc-/- mice (Figure 2E and F) as well as in the BM, PC (Figure 2F) and PBL (data not shown), while the CLL cells of the mb1-CreERT2;Eµ-TCL1 genotype showed massive accumulation in the spleens and also in the BM and PC (Figure 2F) of the recipient Rag2-/-;γc-/- mice leading to their early death. Additionally, the spleens from Rag2-/-;γc-/- mice transplanted with mb1-CreERT2;Igαfl/fl;Eµ-TCL1-derived CLL cells were smaller compared to the control (Online Supplementary Figure S1D and E). Collectively, these findings show that ablation of the BCR in CLL cells is associated with reduced tumor size and increased overall survival demonstrating a clear dependence of CLL cells on BCR.
PI3K activity is reduced in Igα-deficient chronic lymphocytic leukemia cells
PI3K signaling is activated downstream of the BCR and is quintessential for the survival of healthy B cells.25 In order to investigate the effects of BCR deficiency on BCR signaling and the PI3K pathway in CLL cells, we assessed the phosphorylation of the PI3K target AKT and the phosphorylation of other signaling factors downstream of the BCR including LYN, SYK and BTK by intracellular flow cytometry in CLL cells 10 days after induced Igα deletion. The time point of 10 days was selected, because we already observed a complete loss of Igα and BCR expression at this time but enough cells for flow cytometric analysis were still alive. Ten days after induced Igα deletion, the BCR-deficient CLL cells showed decreased AKT phosphorylation at S473 and T308 compared to the BCR-sufficient CLL control cells as well as significantly reduced LYN phosphorylation (Y396/Y507), SYK phosphorylation (Y525/526) and BTK phosphorylation (Y223) (Figure 3A and B). In addition, we analyzed whether the CLL cells could be stimulated by IgM F(ab')2 treatment after Igα deletion. Using a calcium influx assay, we observed that, in contrast to mb1-CreERT2; Eµ-TCL1 CLL control cells, CLL cells from mb1-CreERT2;Igαfl/fl;Eµ-TCL1 mice did not release Ca2+ upon BCR stimulation with IgM F(ab')2 fragments 10 days after Tam treatment (Figure 3E). Moreover, 5 minutes after stimulation with anti-IgM F(ab')2 fragments, the Igα-deficient CLL cells show less increase of SYK-phosphorylation at Y525 and Y526 compared to mb1-CreERT2;Eµ-TCL1 CLL control cells (Online Supplementary Figure S2G).
The decrease in AKT phosphorylation after Igα deletion points to a reduced PI3K activity in the absence of the BCR. Moreover, the decreased phosphorylation of the BCR-proximal kinases LYN, SYK and BTK indicates that BCR signaling is downregulated in CLL B cells in consequence of Igα deletion. Constitutively active PI3K signaling may lead to increased BCL-2 expression.26 Therefore, we investigated the expression of BCL-2 in BCR-deficient and BCR-expressing B cells. BCR-deficient CLL cells from mb1-CreERT2;Igαfl/fl;Eµ-TCL1 had significantly decreased BCL-2 expression 10 days after Tam treatment as compared to control cells (Figure 3C and D). In line with this, we observed a slight downregulation of the anti-apoptotic protein myeloid cell leukemia sequence 1 (MCL-1) in CLL cells 10 days after induced Igα deletion (Online Supplementary Figure S2A and F; left). Considering that BCL-2 is also regulated by NFκB, we tested whether ablation of the BCR in CLL cells from mb1-CreERT2;Igαfl/fl;Eµ-TCL1 mice also resulted in reduced NFκB activity by analyzing IKKα/b phosphorylation at S176/180 and phosphorylation of NFκB p65 at S536 10 days after induced Igα deletion. Interestingly, our analysis revealed that IKKα/b phosphorylation at S176/180, phosphorylation of NFκB p65 at S536 and thus NFκB activity is slightly downregulated in CLL cells with induced Igα deletion compared to Eµ-TCL1 CLL control cells (Online Supplementary Figure S2B, C and F).
Together, these data suggest that BCR-mediated activation of PI3K signaling is essential for the survival of CLL B cells and that reduced NFκB activity and BCL-2 upregulation may be an important part of this regulation.
PTEN-loss augments PI3K activity and results in early onset of chronic lymphocytic leukemia
Next, we explored the role of the PI3K signaling pathway in the onset and maintenance of mouse CLL by inactivating PTEN, the negative regulator of the PI3K signaling pathway. To this end, we generated a mouse model with a B cell-specific and Tam-induced deletion of the Pten gene. In order to investigate the role of PTEN in the onset of mouse-CLL, we crossed the mb1-CreERT2;Eµ-TCL1 mice to the Ptenfl/fl mouse strain to generate mb1-CreERT2;Ptenfl/fl;Eµ-TCL1 mice. These mice were treated with Tam at a young age (6 weeks) before the detection of any CLL B cells in the peripheral blood or the outbreak of mouse CLL that are usually evident at 3 to 6 months in mb1-CreERT2;Ptenfl/fl;Eµ-TCL1 and mb1-CreERT2;Eµ-TCL1 mice, respectively. We assessed the development of CLL cells using the mouse CLL key markers CD19+ B220low CD5+ IgM+ IgD-. mb1-CreERT2;Eµ-TCL1 mice served as controls. Eight and 16 weeks after the beginning of the Tam treatment the mice were analyzed by flow cytometry for the absolute cell number of CD19+ B220low CD5+ IgM+ IgD- B cells. At both time points the number of CLL cells was increased (8 weeks: >2×106, Figure 4A; 16 weeks: >2×107, Figure 4B) in the mb1-CreERT2; Ptenfl/fl;Eµ-TCL1 mice as compared to those from the spleens of the mb1-CreERT2;Eµ-TCL1 control mice. These data show that after loss of PTEN, the resulting constitutive activity of the PI3K pathway leads to accelerated development of CLL in young mice as shown by the accumulation of CLL B cells with time. Recent studies reported that either germline deletion27 or insertion of a single mutation28 in the Tp53 gene (encoding the tumor suppressor p53), a gene, which is often mutated in CLL,29 on Eµ-TCL1 background also accelerated the development of the CLL disease. We deleted the Tp53 gene in mb1-CreERT2;Eµ-TCL1 mice and found out that 24 weeks post treatment 94 % of B cells in the spleens of the mb1-CreERT2;Ptenfl/fl;Eµ-TCL1 mice became CLL cells, whereas the mb1-CreERT2;Tp53fl/fl;Eµ-TCL1 mice showed 2% of CLL cells (Figure 4C). By assessing the expression of p53 by flow cytometry we confirmed that Tp53 was indeed deleted in mb1-CreERT2;Tp53fl/fl;Eµ-TCL1 mice (Figure 4D). In addition, we compared the development of CLL in mice lacking Tp53 expression (mb1-Cre;Tp53fl/fl;Eµ-TCL1) to mice with a constitutive heterozygous loss of Pten (mb1-Cre;Ptenfl/+;Eµ-TCL1). At 32 weeks, the mb1-Cre;Ptenfl/+;Eµ-TCL1 mice showed 94% of CLL cells whereas mb1-Cre;Tp53fl/fl;Eµ-TCL1 mice developed only 27% of CLL B cells in the blood (Figure 4E). Moreover, a combined deletion of Tp53 and Pten in mb1-Cre;Ptenfl/+;Tp53fl/fl;Eµ-TCL1 mice did not accelerate the outbreak of CLL when compared to mb1-Cre;Ptenfl/+;Eµ-TCL1 mice (Online Supplementary Figure S3A). This confirms that the accelerated development of the disease is specifically driven by the partial loss of PTEN and the subsequent activation of the PI3K pathway.
As further signs of elevated PI3K signaling, we found an increased phosphorylation of S473 of AKT and Y223 of BTK in CD19+ B220low CD5+ IgM+ IgD- CLL cells from the spleens of mb1-CreERT2;Ptenfl/fl;Eµ-TCL1 mice compared to CLL cells from the mb1-CreERT2;Eµ-TCL1 control mice (Figure 4F; Online Supplementary Figure S3 D and E). In order to investigate, whether PTEN deficiency resulted in constitutive activation of the PI3K/AKT pathway, we stimulated splenic CLL cells from mb1-CreERT2;Ptenfl/fl;Eµ-TCL1 mice and mb1-CreERT2;Eµ-TCL1 control mice with anti-IgM F(ab')2 fragments and monitored the AKT phosphorylation at S473 and T308 as well as BTK phosphorylation at Y223 after 5 minutes of stimulation. In both cases, we could not observe any significant increase in phosphorylation after BCR stimulation indicating that both, wild-type TCL1 leukemia cells and PTEN-deficient leukemia cells exhibit a constitutive active PI3K/AKT signaling, that cannot be significantly increased by BCR stimulation (Online Supplementary Figure S3B and C).
Additionally, BCL-2 expression was higher in PTEN-deficient CD19+ B220low CD5+ IgM+ IgD- cells relative to the control (Figure 4F, right; Online Supplementary Figure S3F). Statistical analysis confirmed that the increased AKT- and BTK-phosphorylation as well as the increased BCL-2 expression in the PTEN-deficient CLL B cells was significant compared to the PTEN-sufficient control (Figure 4F). Furthermore, we affirmed by flow cytometry that the Pten gene was efficiently deleted in splenic CLL cells from mb1-CreERT2;Ptenfl/fl;Eµ-TCL1 mice and mb1-CreERT2;Eµ-TCL1-derived cells served as a control (Online Supplementary Figure S3G). In conclusion, in the absence of PTEN, the CLL cells exhibit significant increase in AKT and BTK phosphorylation likely leading to higher activity of these molecules. These findings provide evidence for enhanced PI3K signaling in these PTEN-deficient cells. Furthermore, in this model, conditional inactivation of PTEN resulted in the accelerated onset of CLL.
Deletion of Pten leads to autonomous survival of chronic lymphocytic leukemia cells
In order to test the tumorigenic potential of the primary splenic CD19+ B220low CD5+ IgM+ IgD- PTEN-deficient CLL cells from Tam-treated mb1-CreERT2;Ptenfl/fl;Eµ-TCL1 mice, we transferred them into Rag2-/-;γc-/- mice. Two weeks post engraftment, a population of CD19+ B220low CD5+ IgM+ IgD- cells was detected in the spleen (Figure 5A).
Surprisingly, the primary splenic PTEN-deficient CLL cells from Tam-treated mb1-CreERT2;Ptenfl/fl;Eµ-TCL1 mice grew autonomously in culture and preserved the features of mouse CLL cells retaining their CD19+ B220low CD5+ IgM+ IgD- phenotype. The cells were cultured in a medium supplemented only with fetal calf serum and in the absence of any additional growth factors or antigen (Figure 5B). The PTEN deficiency of these cell lines was confirmed by genotyping and flow cytometry (Figure 5C; Online Supplementary Figure S4A, right). We generated five different CLL-like cell lines, which were heterogenous, but grew similarly in culture over prolonged period of time without growth factor supplements.
After growing in culture for 4 months, functional analysis revealed that some of these cell lines mobilized intracellular Ca2+ release upon stimulation with a polyclonal anti-IgM F(ab')2 antibody (Figure 5D). CD19+ B220low CD5+ IgM+ IgD- CLL cells phenotypically resemble B-1 B cells. Since BCR of some B1 B cells are specific to phosphatidyl choline (Ptc), we assessed by flow cytometry whether the CLL-like CreERT2;Ptenfl/fl;Eµ-TCL1 cells were capable of binding Ptc liposomes. We found that some PTEN-deficient CLL-like cell lines could not bind Ptc with their BCR (Online Supplementary Figure S4A, blue line) as compared to PTEN-sufficient CLL-like CreERT2;Eµ-TCL1 control cells (Online Supplementary Figure S4A, red line). In addition, we concluded that PTEN-deficient CLL-like cell lines were polyclonal because we detected multiple Vb-Jb recombination events within one population (Online Supplementary Figure S4B). Taken together, constitutive activation of PI3K signaling through loss of PTEN accelerates CLL development and allows efficient engraftment and maintenance of CLL-like B cell in vivo and in vitro.
Because BCL-2 expression was increased in splenic PTEN-deficient CLL cells from mb1-CreERT2;Ptenfl/fl;Eµ-TCL1 mice, we treated the cells cultured for 4 months with the BCL-2 inhibitor ABT-199 (also known as venetoclax). The relative numbers of the PTEN-deficient as well as PTEN-sufficient cells were 5-fold decreased at a concentration of 10 µM compared to the vehicle (dimethyl sulfoxide [DMSO]) control (Online Supplementary Figure S4C).
Moreover, in order to test whether the PTEN-deficient CLL-like cells in culture were still dependent on BCR signaling for survival and proliferation, we treated the cells with inhibitors blocking BCR downstream signaling and assessed the survival and proliferation of the cells in culture for 4 days. We used LY294002 (10 µM) as a selective PI3K inhibitor, zerumbone (10 µM) to block NFκB activity, ibrutinib (1 µM) to inhibit BTK activity, R406 (5 µM) as a SYK inhibitor and PP2 (10 µM) to block Src-family tyrosine kinases. All of the inhibitors significantly reduced the survival and proliferation of both mb1-CreERT2;Ptend/d;Eµ-TCL1 and mb1-CreERT2;Eµ-TCL1 culture cells when compared to the cells treated with vehicle control (DMSO) (Figure 5E and F). This indicates that the PTEN-deficient cells that are autonomously growing in culture are still dependent on BCR signaling and the activity of NFκB. Interestingly, we observed higher survival and proliferation of Pten-deleted CLL-like cells treated with inhibitors blocking BCR signaling (LY294002, ibrutinib, R406, PP2) when compared to the treated mb1-CreERT2;Eµ-TCL1 leukemic cells (Online Supplementary Figure S4D and E). Vice versa, mb1-CreERT2;Eµ-TCL1 cells treated with the NFκB inhibitor zerumbone showed better survival and proliferation when compared to the PTEN-deficient mb1-CreERT2;Ptend/d;Eµ-TCL1 culture cells (Online Supplementary Figure S4D and E).
In order to test whether the CD19+ B220low CD5+ IgM+ IgD-CLL cells could be generated if Pten was deleted ex vivo on Eµ-TCL1 background, we treated purified primary splenic B2 cells from mb1-CreERT2;Ptenfl/fl;Eµ-TCL1 mice and mb1-CreERT2;Eµ-TCL1 controls with Tam (4-OHT) in culture in the presence of the B-cell activating factor (BAFF). The ex vivo-generated PTEN-deficient CLL cells were more abundant compared to the PTEN-sufficient control (26% to 8%) (Online Supplementary Figure S5A and B). However, these cells could not survive for an extended time period in culture.
Heterozygous loss of Pten accelerates chronic lymphocytic leukemia development
In order to strengthen the evidence that PTEN-deficiency and subsequent enhanced PI3K-activity, monitored by AKT-phosphorylation, lead to accelerated onset of mouse CLL, we intercrossed the mb1-Cre;Eµ-TCL1 with the Ptenfl/+ strains to generate the mb1-Cre; Ptenfl/+;Eµ-TCL1 which allows for a constitutive heterozygous deletion of the Pten gene in B cells. Only one “floxed” Pten allele was introduced because constitutive Pten deletion on both al-leles leads to a block of B-cell development at the pro-B-cell stage due to the inability to express a µHC.30
In these mice, we detected an early development of CLL cells at 8 weeks of age (Figure 6A). This was supported by the quantification of five independent experiments with five individual mice each showing that CLL cells accumulated in the spleens of young (8 weeks old) mb1-Cre;Ptenfl/+;Eµ-TCL1 mice (Figure 6B, blue bar) but not in control mb1-Cre;Eµ-TCL1 mice (Figure 6B, red bar). The CD19+ B220low CD5+ CLL cells expressed only IgM and no IgD (Figure 6C, left) as compared to the CD19+ B220+ CD5-cell population (IgM+ IgD+) from the same mice (Figure 6C, right). Consistent with the heterozygous loss of Pten, the splenic CD19+ B220low CD5+ IgM+ IgD- CLL cells from mb1-Cre;Ptenfl/+;Eµ-TCL1 displayed significantly more AKT phosphorylation than splenic B cells from the mb1-Cre;Eµ-TCL1 mice (Figure 6D and E, left). Together, these results indicate that the constitutive loss of one Pten allele in combination with overexpression of TCL1 significantly accelerates the onset of CLL.
Heterozygous Pten deletion does not lead to Richter’s transformation
It was recently shown by Kohlhaas et al. that constitutive activation of AKT in Eµ-TCL1 mice results in Richter’s transformation (RT), an aggressive lymphoma which occurs upon progression from CLL.31 So, we investigated if mb1-Cre;Ptenfl/+;Eµ-TCL1 mice with a heterozygous Pten deletion develop RT and histologically analyzed spleens from diseased mice for features of RT. RT cells can be distinguished from CLL cells by morphological abnormalities, large lymphoid cells and increased proliferation. Histone H3 phosphorylation on S10 is specific to mitosis and phosphorylated histone H3 (PHH3) proliferation markers are increasingly being used to evaluate proliferation in various tumors.32 Therefore, we analyzed the number of PHH3-positive cells in spleens of diseased mb1-Cre;Ptenfl/+;Eµ-TCL1 and mb1-Cre;Eµ-TCL1 control mice by fluorescence microscopy. However, no significant difference in PHH3 positive cells could be observed (Figure 6F; Online Supplementary Figure S5C) as well as no difference in size of the CLL cells (Online Supplementary Figure S5E). Hematoxylin and eosin (H&E) staining on paraffin embedded splenic sections of diseased mb1-Cre;Ptenfl/+;Eµ-TCL1 and mb1-Cre;Eµ-TCL1 mice also revealed no significant changes after Pten deletion in splenic CLL cells (Online Supplementary Figure S5D). As a second marker for proliferation, we measured Ki-67 levels in splenic B cells of mb1-Cre;Ptenfl/+;Eµ-TCL1 and mb1-Cre;Eµ-TCL1 control mice by flow cytometric analysis. Again, no significant difference in Ki-67 expression levels could be observed (Figure 6E, right; Online Supplementary Figure S5F). So, we assume that loss of PTEN expression does not promote CLL transformation towards RT.
PTEN is downregulated in human chronic lymphocytic leukemia cells
In order to test whether PTEN plays a role in the survival of human CLL, we analyzed its protein expression in B cells from human CLL blood samples. The patient-derived CLL samples were classified according to the presence (mutated, M-CLL) or absence (unmutated, U-CLL) of the mutation in the immunoglobulin heavy chain variable region gene (IGHV). An unmutated IGHV gene is a molecular marker in human CLL associated with poorer prognosis and shorter survival of the patients. As a control we used blood samples from HD (age 60+ years). Overall, we analyzed 14 M-CLL and 21 U-CLL samples as well as 16 HD samples for their PTEN protein expression in B cells using flow cytometry. Statistical analysis revealed a significantly decreased PTEN protein expression in CLL cells when compared to B cells from HD B1 and B2 (Figure 7B and E). In nine of 14 M-CLL (64.3 %) and 17 of 21 U-CLL samples (81 %) there was lower PTEN expression in CLL cells when compared to the mean of the HD B1 control cells (Figure 7B, E and F). The remaining CLL samples showed either similar or slightly higher PTEN protein levels relative to the mean of the HD samples (Figure 7F). As a control, the same samples showed no difference in size or when stained with the secondary Ab alone (Figure 7C). In order to assess whether the downregulation of PTEN protein expression in the 64.3 % M-CLL and 81 % U-CLL samples was controlled in a post-transcriptional or post-translational manner, we measured the PTEN mRNA transcript levels by RT-qPCR. The results revealed that the PTEN mRNA levels were significantly increased in 71.5 % of the M-CLL and in 73.3 % of the U-CLL samples when compared to the HD controls (Figure 7E and F). Therefore, we assumed that the PTEN protein levels in approximately two thirds of the analyzed CLL patients are likely downregulated by either translational repression via microRNA (miRNA) or in a post-translational manner. Indeed, the repression of PTEN transcripts by miRNA has been already reported in many diseases.33,34
Downregulation of PTEN expression by miRNA-21, miRNA-29 and PAX5
Among others, the miRNA miR-21 is known to target the tumor suppressor PTEN as the knockdown of miR-21 in a DLBCL cell line resulted in increased PTEN protein expression but did not affect the level of PTEN mRNA.35 In order to address the miR-21 expression in human CLL, we performed miRNA isolation both from HD, M-CLL and U-CLL patients. The analysis by RT-qPCR revealed a significant increase in the amount of miR-21 in M-CLL and U-CLL B cells compared to HD control B cells (Figure 8A). The miR-29 family of miRNA, consisting of three members miR-29a, miR-29b and miR-29c, is highly expressed in cells of the adaptive immune response and has also been shown to regulate PTEN expression leading to an increased PI3K activity.36 The ablation of miR-29 specifically in B lymphocytes results in an increase in PTEN expression and a decrease of the PI3K activity in mature B cells. We analyzed the expression levels of miR-29a, miR-29b and miR-29c in the HD, M-CLL and U-CLL patients’ samples and could observe a significantly increased expression level of all three miR-29 family members in B cells from both M-CLL and U-CLL patients if compared to the HD controls (Figure 8B). Consequently, the expression of miR-21 and the miR-29 family was shown to be upregulated in human CLL cells, possibly accounting for the reduced PTEN protein levels in these cells.
A recent study has reported that the expression of miR-29 is controlled by PAX5.37 Hence, we analyzed the protein expression of PAX5 in B cells from M-CLL and U-CLL patients as well as HD controls by flow cytometry. Indeed, we could observe significantly higher PAX5 protein expression levels in B cells derived from CLL patients compared to HD (Figure 8C and D, left). As a control, the same samples did not show a difference in size or in the isotype control staining (Figure 8D, right). Together, these data strengthen our hypothesis, that PTEN expression in CLL cells are post-transcriptionally repressed by high levels of the miRNA miR-21 and miR-29, the second upregulated by increased levels of PAX5 itself (Figure 8E).
In this study we present a novel mouse model, which allows the Tamoxifen-inducible inactivation of Igα (subsequently preventing BCR assembly and expression on the cell surface) in a mouse CLL model.
Our findings demonstrate that loss of mature B cells after the ablation of the BCR in combination with the anti-IL-7R treatment is an intrinsic feature of B cells and not due to their reduced production. Consistent with previous findings,25,38-40 we confirm here that mature peripheral B cells rely on their BCR for survival, as almost the complete B cell pool is lost within 2 months after Igα inactivation. We took advantage of the mb1-CreERT2 mouse strain’s efficient recombination of the Igα locus and applied it to the Eµ-TCL1 CLL mouse model, in which we could successfully ablate Igα.
One major finding of this study is that in the Eµ-TCL1 mouse model, the maintenance of CLL cells requires BCR expression because in the absence of Igα 100-fold fewer CLL cells are maintained compared to the Igα-sufficient control. Based on our results, we suggest that in the absence of the BCR PI3K-signaling is reduced, as demonstrated by a decrease in AKT phosphorylation and lower levels of BCL-2 and MCL-1. In addition, the loss of BCR expression in CLL cells results in slightly downmodulated NFκB activity what might additionally contribute to the decreased BCL-2 expression levels. BCL-2 is important for the development and survival of mature naive B cells,41 and its overexpression partially rescues BCR-deficient B cells.42 We further show that ablation of the BCR reduces tumor size and prolongs the overall survival of mice with fully developed CLL.
Srinivasan et al. have shown that BCR-dependent signaling via the PI3K provides the crucial “tonic signal”, which is indispensable for the maintenance of resting mature B cells.25 Although other studies have attributed the micro-environment with a role in CLL development and progres-sion,43 our results demonstrate that, the BCR alone dictates the fate of the CLL cells in the Eµ-TCL1 mouse model by activating the PI3K pathway, the key component of chronic active BCR signaling in mouse CLL.44 This points out the importance of the BCR as a scaffold and platform for signaling emanating from different stimuli. Therefore, our results establish that BCR expression per se is required and indispensable to keep mouse CLL cells alive. To our knowledge, this study provides the first direct genetic evidence that the maintenance of mouse CLL cells depends on the BCR.
The second major finding of this study is that conditional deletion of the Pten gene and subsequent constitutively active PI3K signaling lead to an accelerated onset of CLL development in mice. CLL in Eµ-TCL1 transgenic mice develops after long latencies.20 This indicates that high expression of TCL1 is insufficient to drive transformation and that other genetic or epigenetic changes are presumably required. PTEN deficiency alone does not drive tumorigenesis in mature B cells;26 however, based on our data, inactivation of PTEN with simultaneous overexpression of TCL1 accelerates the onset of CLL pathogenesis in young mice. We show that in PTEN-deficient TCL1-transgenic CLL cells AKT phosphorylation is increased compared to B cells only overexpressing TCL1. As PTEN-deficient splenic CLL cells are not susceptible to BCR stimulation with anti-IgM F(ab’)2 fragments, we believe that PTEN deficiency results in constitutive activation of the PI3K/AKT pathway. However, although Kohlhaas et al. recently reported that constitutive activation of AKT in a Eµ-TCL1 mouse model results in RT31 we were unable to show this phenomenon in Ptenfl/+;mb1-Cre;Eµ-TCL1 mice with heterozygous Pten deletion. These results might be explained by differential progression and development of CLL in the respective mice. While we observe a significantly increased number of CLL cells in Ptenfl/+;mb1-Cre;Eµ-TCL1 mice compared to mb1-Cre;Eµ-TCL1 control mice already at an age of 8 weeks (Figure 6A and B), this difference could not be detected in Eµ-TCL1 mice with constitutive AKT activation before the mice reached an age of 7 months.
Spontaneous apoptosis of CLL cells in vitro has hampered the in-depth investigation of the mechanisms behind CLL maintenance. Cells from spleens of aged mb1-CreERT2;Ptenfl/fl;Eµ-TCL1 mice with high tumor load proliferated in culture without addition of growth factors. Flow cytometric analysis showed that the cells maintained the phenotype of the primary leukemia even after prolonged in vitro culture. To the best of our knowledge, Eµ-TCL1 leukemia-derived cell lines have not been described to date. Recently, a similar phenomenon was observed by Chakraborty et al. wherein murine Eµ-TCL1 leukemia cells exhibiting biallelic inactivation of TP53, CDKN2A and CDKN2B were also found to proliferate spontaneously in vitro.39 Notably, cell lines from mb1-CreERT2;Ptenfl/fl;Eµ-TCL1 were as susceptible to venetoclax as control cells from the mb1-CreERT2;Eµ-TCL1 mice, showing that the loss of PTEN does not confer resistance to apoptosis. Moreover, the PTEN-deficient CLL-like culture cells still relied on BCR signals for proliferation as BCR signaling inhibitors caused decreased survival and proliferation of the cells in vitro. Interestingly, significantly increased cell death and decreased proliferation of PTEN-deficient mb1-CreERT2;Ptend/d;Eµ-TCL1 culture cells in comparison to mb1-CreERT2;Eµ-TCL1 leukemic cells could be observed due to NFκB inhibitor treatment, indicating that the NFκB pathway plays an important role in these PTEN-deficient CLL-like culture cells.
These PTEN-deficient CLL-like cell lines may be well suited for high-throughput screening of novel compounds for CLL treatment. Furthermore, the PTEN-deficient cells can be transplanted into immunodeficient mice and may be used in further in vivo studies. The rapid development of CLL in these mice may help to dissect signaling mechanisms of CLL cells within a reasonable time frame in contrast to the slow disease development in Eµ-TCL1 mice. Therefore, this specifically provides a tool to develop novel treatment options for drug-resistant CLL.
It is remarkable that although splenic CLL cells from mb1-CreERT2;Ptenfl/fl;Eµ-TCL1 mice developed in culture after ex vivo deletion of Pten, they were not immortalized like the cells, in which Pten had been deleted in vivo. This may suggest that additional factors or additive mutations are required to promote transformation in vivo.
PTEN is tightly regulated by various non-genomic mechanisms including epigenetic silencing, post-transcriptional regulation by non-coding RNA, and post-translational modification.45 Due to the high PTEN mRNA transcript levels in the analyzed human CLL samples which stand in contrast to the decreased PTEN protein expression, we assume that the PTEN downregulation in two thirds of overall 35 analyzed CLL samples might be regulated in a post-transcriptional manner mediated by non-coding RNA. miRNA comprise a large family of small non-coding RNA that emerged as post-transcriptional regulators of gene expression.46 The microRNA miR-21, miR-155, miR-17-92 or miR-19 and miR-29, for instance, are post-transcriptional regulators of PTEN expression, which directly target PTEN and contribute to its reduced expression in CLL.47 Moreover, several studies demonstrated that microRNA expression profiles can be used to distinguish normal B cells from malignant CLL cells and that miRNA signatures are associated with prognosis and progression of CLL.48 Among other miRNA that have also been shown to regulate PTEN expression the miR-29 family is one of the critical miRNA that play a role in cancer pathogenesis.49 It was revealed that Eµ-miR-29 transgenic mice overexpressing miR-29 in B cells exhibit an expanded CD5+ B-cell population with 20% of the mice developed leukemia indicating a role of miR-29 in the pathogenesis of B-CLL.50 Our analysis of miR-29 expression in human CLL cells revealed that all three miR-29 family miRNA were significantly over-expressed in both M-CLL and U-CLL patient samples. Moreover, we could show that PAX5 expression is upregulated in human CLL patient samples, which might induce the upregulation of the miR-29 family miRNA. This stands in line with the recently published findings of Calderón et al. who identified PAX5 as an enhancer of PI3K signaling that downregulates PTEN expression in mature B cells, likely by controlling the abundance of PTEN-targeting miRNA.37
In summary, our data illustrate that the loss of PTEN in murine CLL cells results in the accelerated development of the disease. This observation underscores the significance of PI3K signaling in the pathogenesis of CLL. In addition, the significant downregulation of PTEN expression in B cells of around two third of the analyzed CLL patients suggests an important role of PTEN in the development and maintenance of human CLL. It is conceivable that the decreased PTEN expression is induced by the increased levels of miR-21, miR-29 family and PAX5 expression in the analyzed human CLL cells as PAX5 is believed to restrain PTEN expression in B cells by controlling the expression of PTEN-targeting miRNA.37 Conclusively, the work described in this paper strengthens the important role of PTEN as a tumor suppressor in CLL and raises a number of interesting questions that may help to design the potential use of PTEN-targeting miRNA inhibitor strategies for CLL.
Furthermore, we show that BCR loss fully abrogates the survival of CLL cells in mice. We therefore conclude that PTEN expression sets a threshold for malignant transformation in the presence of BCR. Targeting the BCR itself, however, may be a major future achievement in combatting CLL, since antibodies against Ig(3 have been shown to be potent in depleting autoimmune and malignant B cells in mouse models and in preclinical studies.
- Received September 1, 2021
- Accepted January 4, 2022
No conflicts of interest to disclose.
VS, AK, NA, and EH performed experiments and analyzed data. LN provided the IgaTMF mice; KR provided ES cells targeted with IgaTMF and discussed the study; EH and HJ designed the study and proposed the experiments; EH supervised the work and wrote the manuscript with VS. All the authors read the manuscript and discussed the results.
This work was supported by the DFG through SFB1074 (Experimental Models and Clinical Translation in Leukemia) projects A9, A10 and through TRR130 (B cells: Immunity and Autoimmunity) projects P01, P02, P04, P08 and C03, and through EXC294, and ERC advanced grants 694992 to HJ.
We thank Duygu Yağdıran, Karoline Lodd, Katharina Goehring, Lisa Gögler, Selina Fahrenholz, Stefanie Brey and Andrea Schneider for their technical support and Ella Levit-Zerdoun for proof-reading the manuscript.
- Li Y, Wang Y, Wang Z, Yi D, Ma S.. Racial differences in three major NHL subtypes: descriptive epidemiology. Cancer Epidemiol. 2015; 39(1):8-13. https://doi.org/10.1016/j.canep.2014.12.001PubMedPubMed CentralGoogle Scholar
- Stevenson FK, Krysov S, Davies AJ, Steele AJ, Packham G.. B-cell receptor signaling in chronic lymphocytic leukemia. Blood. 2011; 118(16):4313-4320. https://doi.org/10.1182/blood-2011-06-338855PubMedGoogle Scholar
- Seda V, Mraz M.. B-cell receptor signalling and its crosstalk with other pathways in normal and malignant cells. Eur J Haematol. 2015; 94(3):193-205. https://doi.org/10.1111/ejh.12427PubMedGoogle Scholar
- Damle RN, Wasil T, Fais F. Ig V gene mutation status and CD38 expression as novel prognostic indicators in chronic lymphocytic leukemia. Blood. 1999; 94(6):1840-1847. https://doi.org/10.1182/blood.V94.6.1840PubMedGoogle Scholar
- Hamblin TJ, Davis Z, Gardiner A, Oscier DG, Stevenson FK. Unmutated Ig V(H) genes are associated with a more aggressive form of chronic lymphocytic leukemia. Blood. 1999; 94(6):1848-1854. https://doi.org/10.1182/blood.V94.6.1848PubMedGoogle Scholar
- Stamatopoulos K, Agathangelidis A, Rosenquist R, Ghia P.. Antigen receptor stereotypy in chronic lymphocytic leukemia. Leukemia. 2017; 31(2):282-291. https://doi.org/10.1038/leu.2016.322PubMedGoogle Scholar
- Philippen A, Diener S, Zenz T, Dohner H, Stilgenbauer S, Mertens D.. SYK carries no activating point mutations in patients with chronic lymphocytic leukaemia (CLL). Br J Haematol. 2010; 150(5):633-636. https://doi.org/10.1111/j.1365-2141.2010.08244.xPubMedGoogle Scholar
- Duhren-von Minden M, Ubelhart R, Schneider D. Chronic lymphocytic leukaemia is driven by antigen-independent cell-autonomous signalling. Nature. 2012; 489(7415):309-312. https://doi.org/10.1038/nature11309PubMedGoogle Scholar
- Patel V, Balakrishnan K, Bibikova E. Comparison of acalabrutinib, a selective Bruton tyrosine kinase inhibitor, with ibrutinib in chronic lymphocytic leukemia cells. Clin Cancer Res. 2017; 23(14):3734-3743. https://doi.org/10.1158/1078-0432.CCR-16-1446PubMedPubMed CentralGoogle Scholar
- Woyach JA, Bojnik E, Ruppert AS. Bruton's tyrosine kinase (BTK) function is important to the development and expansion of chronic lymphocytic leukemia (CLL). Blood. 2014; 123(8):1207-1213. https://doi.org/10.1182/blood-2013-07-515361PubMedPubMed CentralGoogle Scholar
- Burger JA, Chiorazzi N.. B cell receptor signaling in chronic lymphocytic leukemia. Trends Immunol. 2013; 34(12):592-601. https://doi.org/10.1016/j.it.2013.07.002PubMedPubMed CentralGoogle Scholar
- Ten Hacken E, Burger JA. Molecular pathways: targeting the microenvironment in chronic lymphocytic leukemia - focus on the B-cell receptor. Clin Cancer Res. 2014; 20(3):548-556. https://doi.org/10.1158/1078-0432.CCR-13-0226PubMedGoogle Scholar
- Werner M, Hobeika E, Jumaa H.. Role of PI3K in the generation and survival of B cells. Immunol Rev. 2010; 237(1):55-71. https://doi.org/10.1111/j.1600-065X.2010.00934.xPubMedGoogle Scholar
- Okkenhaug K, Vanhaesebroeck B.. PI3K in lymphocyte development, differentiation and activation. Nat Rev Immunol. 2003; 3(4):317-330. https://doi.org/10.1038/nri1056PubMedGoogle Scholar
- Downes CP, Ross S, Maccario H, Perera N, Davidson L, Leslie NR. Stimulation of PI 3-kinase signaling via inhibition of the tumor suppressor phosphatase, PTEN. Adv Enzyme Regul. 2007; 47:184-194. https://doi.org/10.1016/j.advenzreg.2006.12.018PubMedGoogle Scholar
- Thick J, Metcalfe JA, Mak YF. Expression of either the TCL1 oncogene, or transcripts from its homologue MTCP1/c6.1B, in leukaemic and non-leukaemic T cells from ataxia telangiectasia patients. Oncogene. 1996; 12(2):379-386. Google Scholar
- Herling M, Patel KA, Khalili J. TCL1 shows a regulated expression pattern in chronic lymphocytic leukemia that correlates with molecular subtypes and proliferative state. Leukemia. 2006; 20(2):280-285. https://doi.org/10.1038/sj.leu.2404017PubMedGoogle Scholar
- Chen SS, Chiorazzi N.. Murine genetically engineered and human xenograft models of chronic lymphocytic leukemia. Semin Hematol. 2014; 51(3):188-205. https://doi.org/10.1053/j.seminhematol.2014.05.001PubMedGoogle Scholar
- Bresin A, D'Abundo L, Narducci MG. TCL1 transgenic mouse model as a tool for the study of therapeutic targets and microenvironment in human B-cell chronic lymphocytic leukemia. Cell Death Dis. 2016; 7(1):e2071. https://doi.org/10.1038/cddis.2015.419PubMedPubMed CentralGoogle Scholar
- Bichi R, Shinton SA, Martin ES. Human chronic lymphocytic leukemia modeled in mouse by targeted TCL1 expression. Proc Natl Acad Sci U S A. 2002; 99(10):6955-6960. https://doi.org/10.1073/pnas.102181599PubMedPubMed CentralGoogle Scholar
- Kraus M, Alimzhanov MB, Rajewsky N, Rajewsky K.. Survival of resting mature B lymphocytes depends on BCR signaling via the Igalpha/beta heterodimer. Cell. 2004; 117(6):787-800. https://doi.org/10.1016/j.cell.2004.05.014PubMedGoogle Scholar
- Hobeika E, Levit-Zerdoun E, Anastasopoulou V. CD19 and BAFF-R can signal to promote B-cell survival in the absence of Syk. EMBO J. 2015; 34(7):925-939. https://doi.org/10.15252/embj.201489732PubMedPubMed CentralGoogle Scholar
- Suzuki A, Yamaguchi MT, Ohteki T. T cell-specific loss of Pten leads to defects in central and peripheral tolerance. Immunity. 2001; 14(5):523-534. https://doi.org/10.1016/S1074-7613(01)00134-0Google Scholar
- Hobeika E, Thiemann S, Storch B. Testing gene function early in the B cell lineage in mb1-cre mice. Proc Natl Acad Sci U S A. 2006; 103(37):13789-13794. https://doi.org/10.1073/pnas.0605944103PubMedPubMed CentralGoogle Scholar
- Srinivasan L, Sasaki Y, Calado DP. PI3 kinase signals BCR-dependent mature B cell survival. Cell. 2009; 139(3):573-586. https://doi.org/10.1016/j.cell.2009.08.041PubMedPubMed CentralGoogle Scholar
- Setz CS, Hug E, Khadour A. PI3K-mediated Blimp-1 activation controls B Cell selection and homeostasis. Cell Rep. 2018; 24(2):391-405. https://doi.org/10.1016/j.celrep.2018.06.035PubMedPubMed CentralGoogle Scholar
- Liu J, Chen G, Feng L. Loss of p53 and altered miR15-a/16-1short right arrowMCL-1 pathway in CLL: insights from TCL1-Tg:p53(-/-) mouse model and primary human leukemia cells. Leukemia. 2014; 28(1):118-128. https://doi.org/10.1038/leu.2013.125PubMedPubMed CentralGoogle Scholar
- Lee HJ, Gallardo M, Ma H. p53-independent ibrutinib responses in an Emu-TCL1 mouse model demonstrates efficacy in high-risk CLL. Blood Cancer J. 2016; 6(6):e434. https://doi.org/10.1038/bcj.2016.41PubMedPubMed CentralGoogle Scholar
- Landau DA, Tausch E, Taylor-Weiner AN. Mutations driving CLL and their evolution in progression and relapse. Nature. 2015; 526(7574):525-530. https://doi.org/10.1038/nature15395PubMedPubMed CentralGoogle Scholar
- Alkhatib A, Werner M, Hug E. FoxO1 induces Ikaros splicing to promote immunoglobulin gene recombination. J Exp Med. 2012; 209(2):395-406. https://doi.org/10.1084/jem.20110216PubMedPubMed CentralGoogle Scholar
- Kohlhaas V, Blakemore SJ, Al-Maari M. Active Akt signaling triggers CLL toward Richter transformation via overactivation of Notch1. Blood. 2021; 137(5):646-660. https://doi.org/10.1182/blood.2020005734PubMedGoogle Scholar
- Kim JY, Jeong HS, Chung T. The value of phosphohistone H3 as a proliferation marker for evaluating invasive breast cancers: a comparative study with Ki67. Oncotarget. 2017; 8(39):65064-65076. https://doi.org/10.18632/oncotarget.17775PubMedPubMed CentralGoogle Scholar
- Sandhu SK, Fassan M, Volinia S. B-cell malignancies in microRNA Emu-miR-17~92 transgenic mice. Proc Natl Acad Sci U S A. 2013; 110(45):18208-18213. https://doi.org/10.1073/pnas.1315365110PubMedPubMed CentralGoogle Scholar
- Battistella M, Romero M, Castro-Vega LJ. The high expression of the microRNA 17-92 cluster and its paralogs, and the downregulation of the target gene PTEN, is associated with primary cutaneous B-cell lymphoma progression. J Invest Dermatol. 2015; 135(6):1659-1667. https://doi.org/10.1038/jid.2015.27PubMedGoogle Scholar
- Bai H, Wei J, Deng C, Yang X, Wang C, Xu R.. MicroRNA-21 regulates the sensitivity of diffuse large B-cell lymphoma cells to the CHOP chemotherapy regimen. Int J Hematol. 2013; 97(2):223-231. https://doi.org/10.1007/s12185-012-1256-xPubMedGoogle Scholar
- Hines MJ, Coffre M, Mudianto T. miR-29 sustains B cell survival and controls terminal differentiation via regulation of PI3K signaling. Cell Rep. 2020; 33(9):108436. https://doi.org/10.1016/j.celrep.2020.108436PubMedPubMed CentralGoogle Scholar
- Calderon L, Schindler K, Malin SG. Pax5 regulates B cell immunity by promoting PI3K signaling via PTEN down-regulation. Sci Immunol. 2021; 6(61):eabg5003. https://doi.org/10.1126/sciimmunol.abg5003PubMedPubMed CentralGoogle Scholar
- Levit-Zerdoun E, Becker M, Pohlmeyer R. Survival of Igalpha-deficient mature B cells requires BAFF-R function. J Immunol. 2016; 196(5):2348-2360. https://doi.org/10.4049/jimmunol.1501707PubMedGoogle Scholar
- Chakraborty S, Martines C, Porro F. B-cell receptor signaling and genetic lesions in TP53 and CDKN2A/CDKN2B cooperate in Richter transformation. Blood. 2021; 138(12):1053-1066. https://doi.org/10.1182/blood.2020008276PubMedGoogle Scholar
- Iacovelli S, Hug E, Bennardo S. Two types of BCR interactions are positively selected during leukemia development in the Eµ-TCL1 transgenic mouse model of CLL. Blood. 2015; 125(10):1578-1588. https://doi.org/10.1182/blood-2014-07-587790PubMedPubMed CentralGoogle Scholar
- Nakayama K, Nakayama K, Negishi I. Disappearance of the lymphoid system in Bcl-2 homozygous mutant chimeric mice. Science. 1993; 261(5128):1584-1588. https://doi.org/10.1126/science.8372353PubMedGoogle Scholar
- Lam KP, Kuhn R, Rajewsky K.. In vivo ablation of surface immunoglobulin on mature B cells by inducible gene targeting results in rapid cell death. Cell. 1997; 90(6):1073-1083. https://doi.org/10.1016/S0092-8674(00)80373-6Google Scholar
- Ten Hacken E, Burger JA. Microenvironment interactions and B-cell receptor signaling in chronic lymphocytic leukemia: implications for disease pathogenesis and treatment. Biochim Biophys Acta. 2016; 1863(3):401-413. https://doi.org/10.1016/j.bbamcr.2015.07.009PubMedPubMed CentralGoogle Scholar
- Brown JR. The PI3K pathway: clinical inhibition in chronic lymphocytic leukemia. Semin Oncol. 2016; 43(2):260-264. https://doi.org/10.1053/j.seminoncol.2016.02.004PubMedGoogle Scholar
- Song MS, Salmena L, Pandolfi PP. The functions and regulation of the PTEN tumour suppressor. Nat Rev Mol Cell Biol. 2012; 13(5):283-296. https://doi.org/10.1038/nrm3330PubMedGoogle Scholar
- Bartel DP. MicroRNAs: target recognition and regulatory functions. Cell. 2009; 136(2):215-233. https://doi.org/10.1016/j.cell.2009.01.002PubMedPubMed CentralGoogle Scholar
- Musilova K, Mraz M.. MicroRNAs in B-cell lymphomas: how a complex biology gets more complex. Leukemia. 2015; 29(5):1004-1017. https://doi.org/10.1038/leu.2014.351PubMedGoogle Scholar
- Calin GA, Liu CG, Sevignani C. MicroRNA profiling reveals distinct signatures in B cell chronic lymphocytic leukemias. Proc Natl Acad Sci U S A. 2004; 101(32):11755-11760. https://doi.org/10.1073/pnas.0404432101PubMedPubMed CentralGoogle Scholar
- Pekarsky Y, Croce CM. Is miR-29 an oncogene or tumor suppressor in CLL?. Oncotarget. 2010; 1(3):224-227. https://doi.org/10.18632/oncotarget.129PubMedPubMed CentralGoogle Scholar
- Santanam U, Zanesi N, Efanov A. Chronic lymphocytic leukemia modeled in mouse by targeted miR-29 expression. Proc Natl Acad Sci U S A. 2010; 107(27):12210-12215. https://doi.org/10.1073/pnas.1007186107PubMedPubMed CentralGoogle Scholar
Figures & Tables
This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.