Chronic myeloid leukemia (CML) is a clonal myeloproliferative disorder, arising from a hematopoietic stem cell (HSC) that acquires the chromosomal translocation t(9;22), resulting in the BCR-ABL oncoprotein. While BCR-ABL-targeting tyrosine kinase inhibitors (TKI) eliminate the majority of CML cells, the most primitive disease-initiating leukemic stem cells (LSC) are frequently spared. CML development is accompanied by increasing levels of inflammatory cytokines, such as IL-1α, IL-1β, IL-6, or TNF. Correspondingly, LSC are characterized by a TKI-persisting, inflammatory cytokine response pattern that was observed even during prolonged therapy.1 Our preliminary work confirmed that TNF signaling is active in murine CML stem cells and TNF-targeted antibody treatment enhanced therapeutic efficiency of TKI treatment.2 Moreover IKK-dependent activation of NF-kB has been shown to contribute to BCR-ABL-induced myeloid and lymphoid leukemogenesis.3,4 In CML stem and progenitor cells, an autocrine TNF loop and NF-kB signaling were found to persist during TKI treatment in vitro.5 NF-kB serves as a central inflammatory hub as several cytokines induce IKK-dependent phosphorylation at serine 32/36 and subsequent degradation of IkBα, which releases the NF-kB complex, allowing its activation by phosphorylation at serine 536 and transport to the nucleus.6 Thereby, NF-kB induces transcription of target genes such as several cytokines or the NF-kB signaling molecules themselves.7 Here we confirmed TKI-persistent TNF-induced NF-kB signaling in CML mice and human cells. As TNF can induce pro-proliferative signaling via NF-kB but also pro-apoptotic cascades via RIPK1-induced CASP8 activation, we here aimed to specifically inhibit the pro-proliferative activity by targeting IKK2. Therefore, we performed pharmacologic IKK2 inhibition, using the small molecule IKK2 inhibitor LY2409881 (LY), previously shown to be well tolerable in pre-clinical application.8 Combined BCR-ABL/IKK2-targeting blocked malignant NF-kB signaling and enhanced apoptosis in TKI-sensitive but also TKI-resistant cells. In vivo, NF-kB-mediated TNF activity was elevated despite nilotinib (NIL) treatment. IKK2 targeting severely reduced LSC, which was further reflected by preventing disease onset upon secondary transplantation. As TNF-induced NF-kB-signaling was evident in chronicphase (CP) but likewise blast crisis (BC) CML, IKK2 targeting affected the clonogenic potential in both disease stages. Finally, in CP CML CD34+ cells, combined BCR-ABL/IKK2 inhibition significantly induced apoptosis, also in quiescent LSC showing that this approach enables the eradication of the TKI-persisting malignant stem cell population in CML.
Aiming to get insight into the role of malignant TNF signaling in CML, we started to compare BCR-ABL-positive with ABL-negative cell lines. We observed significant up-regulation of TNF signaling in 32D BCR-ABL versus empty vector control cells, as represented by increased expression of relevant TNF targets in BCR-ABL positive cells (IkBα, fold change (fc): 1.9; Nf-kb, fc: 2.2, A20, fc: 6.5; Online Supplementary Figure S1A). In order to mimic the malignant inflammatory niche, TNF was subsequently added to both cell types at a physiological concentration and this still resulted in increased TNF target gene expression in BCR-ABL versus control cells (IkBα, fc: 1.7; Nf-kb, fc: 3, A20, fc: 3.4). Notably, this upregulation of TNF signaling persisted in the presence of NIL (Online Supplementary Figure S1A). TKI-persistent NF-kB activation was likewise evident in vivo, as shown by bone marrow (BM) immunoh-istochemical p65 staining of Scl-tTa-BCR-ABL or Scl-tTa control mice (Figure 1A). Although NIL slightly reduced p65 protein expression, reversion to healthy control levels was not achieved.
Next, we studied the effects induced by pharmacologic inhibition of the IkB-kinase-subunit, IKK2 using LY. The small molecule inhibitor prevents IKK2 phosphorylation and subsequent degradation of IkBα, which thus remains bound to the NF-kB complex to block its phosphorylation. This prevents nuclear translocation of NF-kB and thereby expression of target genes. Analysis of A20 expression in human KCL22 CML cells suggested a NIL-induced upregulation (Figure 1B; fc: 3.37), that was reduced by LY treatment (A20, fc: 2.02). We observed similar effects when adding imatinib (IM) or dasatinib (DAS) (Online Supplementary Figure S1B). We studied signaling activity via western blot analyses in KCL-22 cells (Figure 1C). As expected, phosphorylation of NF-kB subunit p65 was not reduced by NIL treatment alone. Moreover, pIkBα at serine 32/36 was significantly inhibited by LY single treatment (fc: 0.27) and this was still achieved in the presence of NIL (fc: 0.17), as shown by significant downregulation as compared to the corresponding DMSO control or NIL single treatment, in the presence of TNF. This further resulted in reduced phosphorylation of p65 at serine 536 in the presence of LY (fc: 0.42) or both drugs (fc: 0.29), as compared to the corresponding DMSO control.
Annexin V staining of treated KCL-22 cells showed the advantage of IkB kinase inhibition as this induced a significant increase in apoptosis by IKK2 inhibition alone (fc: 2.4) that further enhanced NIL-induced cell death by 1.4-fold (Figure 1D). The therapeutic effect of IKK2 inhibition was also evaluated in the context of other clinically relevant TKI. For all TKI tested, we observed increased apoptosis levels upon combined treatment (DAS fc: 1.3, BOS fc: 1.4, PON fc: 1.3) (Figure 1D). We next treated isolated lin- BM cells from Scl-tTa-BCR-ABL or Scl-tTa control mice (Figure 1E), showing that control cells remained largely unaffected whereas apoptosis was induced upon LY monotherapy (fc: 2.2) in murine primary CML cells. Moreover, combined LY and NIL treatment enhanced NIL-induced apoptosis by 2.1-fold.
In order to study the role of TNF signaling in BCR-ABL ki-nase-mutated TKI resistance, we utilized the KCL-22Y272H9 and KCL-22T315I-cell lines.10 In KCL-22Y272H cells, LY therapy induced apoptosis, while NIL alone had no effect (Figure 1F). As DAS, bosutinib (BOS), and ponatinib (PON) could bypass the P-loop mutation, these TKI indeed induced KCL-22Y272H cell death that was further elevated with IKK2 inhibition, as reflected by a further rise in Annexin V positivity (DAS fc: 1.4, BOS fc: 1.3, PON fc: 1.5). In KCL-22T315I cells, effects were very modest with LY application only slightly inducing apoptosis and this was unaffected by TKI treatment (Online Supplementary Figure S1C).
In order to investigate whether IKK2 targeting could affect TKI-persistent LSC, we implemented the Scl-tTa-BCR-ABL mouse model (Figure 2A). As expected, splenomegaly and white blood cell count were normalized by NIL treatment (Online Supplementary Figure S2A, B). Comprehensive immunophenotyping revealed a predominant expansion of malignant B cells (B220low) that responded to BCR-ABL inhibition (Online Supplementary Figure S2C, D). Moreover, we confirmed our previous findings on reduced BM-derived long-term (LT)-HSC (Lin-, Sca1+, c-kit+, CD48low, CD150+) upon disease development (Figure 2B).11 In line with the concept that TKI treatment selects for primitive LSC, we observed increased LT-HSC in NIL- or NIL/LY-treated mice. Interestingly, LY monotherapy decreased LT-HSC by 2.3-fold. While Tnf expression was decreased upon treatment in BM, NIL monotherapy, as well as combined NIL/LY therapy apparently increased Tnf transcript levels in the spleen (Figure 2C). As TNF acts both, autocrine and paracrine, this likely resulted in elevated A20 expression in both organs (Figure 2C). In light of this, A20 is not only described to be increased in response to TNF, but also known to protect cells from TNF–induced apoptosis.12 Treatment efficacy was further shown by a reduction of BCR-ABL expression, with the strongest reduction upon combined BCR-ABL- and IKK2 inhibtion (Figure 2D). We assessed the malignant stem cell potential via serial transplantation of BM cells into irradiated wild-type recipients (Figure 2A). In line with our finding that BM-derived LT-HSC are spared by TKI or TKI + LY treatment, secondary recipients showed disease recurrence, accompanied by slightly increased spleen weights (Online Supplementary Figure S2E), as well as elevated BCR-ABL transcript levels (Figure 2E). Interestingly, LY monotherapy prevented spleen expansion and severely impaired re-expansion of malignant cells as shown by only residual BCR-ABL transcript levels, in secondary recipients.
In order to determine whether TNF-signaling is specifically active in LSC, we re-analyzed microarray data (GSE40721) revealing upregulation of TNF signaling via NF-kB in CD34+CD38- stem cells versus CD34+CD38+ progenitor cells (Figure 3A). We proceeded to evaluate the clonogenic potential in CML CD34+ cells upon treatment and this was strongly reduced by both single treatments, as compared to DMSO control (LY: 12.1-fold, NIL: 3.8-fold) and almost completely abrogated upon combined IKK2 and BCR-ABL targeting (77.5-fold; Figure 3B). In order to assess the effect on most primitive cells, replating was performed without any further treatment and this showed that previous NIL treatment had spared malignant stem cells while LY-treated cells were severely impaired in the ability to form colonies (Figure 3C), suggesting that LY monotherapy targets the most primitive malignant stem cells. In healthy donor-derived CD34+ cells, IKK2 inhibition showed an effect in two of six samples (Online Supplementary Figure S3A). As these cells were partially provided by osteoarthritis patients, we assume that IKK2 inhibition could affect these samples, as pro-inflammatory cytokines could be likewise involved.13 In order to dissect if IKK2 targeting induces apoptosis in the most primitive CML cells we performed Annexin V staining in CD34+/CFSEMAX quiescent LSC. Indeed, in vitro apoptosis was significantly increased by IKK2 inhibition alone (fc: 7.4) or in combination with NIL (fc: 8.5; Figure 3D), suggesting that LY treatment exerts its anti-leukemic effects observed upon secondary transplantation at least partially by induction of apoptosis within the LSC compartment.
As further LSC persistence-mediating cytokines activate the NF-kB pathway, we next tested the treatment effect in the presence of physiological IL-1α or IL-1b levels (Online Supplementary Figure S3B). Again, colony formation was significantly reduced by inhibition of IKK2 alone (IL-1α: 1.5fold, IL-1b: 1.7-fold) as well as by combined IKK2- and BCR-ABL-targeting (IL-1α: 3.4-fold, IL-1b: 5.2-fold), suggesting that IKK2 targeting could exerts its effects by inhibiting, not only TNF-mediated NF-kB activation but also due to targeting further pro-leukemic inflammatory signaling.
In the context of progressed disease, IKK2 was described as a resistance-associated gene.14
In line with this, TNF signaling increased in BM CD34+ cells upon CML progression (Figure 3E). Therefore, we studied IKK2 targeting also in primary BC-CML samples, showing that impaired colony formation was evident upon LY exposure in all patient samples tested (CML-BC #1: 10.9-fold; #2: 7.4-fold; #3: 13.9-fold; Figure 3F; Online Supplementary Figure S3C). Again, in combination this was further reduced by NIL (CML-BC #1: 21.4-fold; #2: 42-fold; #3: 93.7-fold). In conclusion, our results underline the potential of NF-kB targeting, via IKK2-inhibition, to eliminate TKI-persisting LSC. In vivo treatment revealed the complexity. While NIL alone and in combination was able to reduce leukemic cells, LSC persisted and re-initiated the disease upon secondary transplant. Treatment with NIL expanded CD3+ T cells, which was still observed when combined with LY but not in LY mono-therapy-treated mice (Online Supplementary Figure S2C). As T cells in CML display a TNF-dominated cytokine secretion profile,15 we assume this could be a source of enhanced splenic Tnf expression, probably exerting paracrine effects on the BM, thereby potentially protecting BM-residing LSC.
- Received March 10, 2022
- Accepted November 23, 2022
No conflicts of interest to disclose.
MB designed research, performed experiments, analyzed the data and wrote the manuscript; FJT and JB performed experiments and revised the manuscript; NC, TJ and ST assisted experimental work and revised the manuscript; IGC and TM reanalyzed publicly available expression data sets; MC contributed patient samples and clinical data; THB contributed research material and revised the manuscript; MS designed research, analyzed the data and revised the manuscript. Final approval of the manuscript was provided by all authors.
All technical information pertaining to the experimentation performed is available on request.
See also: [GSE40721]
The authors thank Dr. Vignir Helgason and Dr. Eric Kalkman for providing KCL22 T315I cells, Julia Plum and Kristina Pannen for excellent technical assistance, and Dr. Jörg Eschweiler for providing healthy donor-derived cells. This work was supported by the IHC (immunohistochemistry) facility, a core facility of the Interdisciplinary Center for Clinical Research (IZKF) Aachen, within the Faculty of Medicine at RWTH Aachen University. Figures were created with BioRender.com.
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