Treatment for myelodysplastic syndromes (MDS) remains insufficient due to clonal heterogeneity and lack of effective clinical therapies. Dysregulation of apoptosis is observed across MDS subtypes regardless of mutations and represents an attractive therapeutic opportunity. Venetoclax (VEN), a selective inhibitor of anti-apoptotic protein B-cell lymphoma- 2 (BCL2), has yielded impressive responses in older patients with acute myeloid leukemia (AML) and high risk MDS. BCL2 family anti-apoptotic proteins BCL-XL and induced myeloid cell leukemia 1 (MCL1) are implicated in leukemia survival, and upregulation of MCL1 is seen in VEN-resistant AML and MDS. We determined in vitro sensitivity of MDS patient samples to selective inhibitors of BCL2, BCL-XL and MCL1. While VEN response positively correlated with MDS with excess blasts, all MDS subtypes responded to MCL1 inhibition. Treatment with combined VEN + MCL1 inhibtion was synergistic in all MDS subtypes without significant injury to normal hematopoiesis and reduced MDS engraftment in MISTRG6 mice, supporting the pursuit of clinical trials with combined BCL2 + MCL1 inhibition in MDS.
Myelodysplastic syndromes (MDS) are heterogeneous bone marrow failure neoplasms marked by cytopenias, reduced quality of life and predilection to transform into acute myeloid leukemia (AML). Readily available treatments for MDS are lacking, and adapting newly approved therapies designed for AML to MDS is complicated, largely due to the heterogeneity of MDS. Despite this, and consistent with other myeloid malignancies, clonal hematopoietic stem and progenitor cells (HSPC) in MDS avoid programmed cell death often and share an imbalanced pattern of mitochondrial-controlled BCL2 family proteins.1-4 The BCL2 family includes anti-apoptotic and pro-apoptotic proteins that compete for ligand to block or promote the activation of BAX/BAK oligomerization that is required to induce mitochondrial outer membrane potential and subsequent apoptosis.5,6 As a method to evade apoptosis, cancer cells often upregulate anti-apoptotic proteins BCL2 and MCL1, leading to a survival advantage in numerous malignancies.5-7 Thus, selective targeting of anti-apoptotic proteins is a viable treatment strategy. Venetoclax (VEN), a Food and Drug Administration-approved therapy that specifically inhibits the anti-apoptotic protein, BCL2, has yielded response rates of up to 50-70% in elderly AML when combined with DNA methyltransferase inhibitors (DNMTi) or low dose cytarabine,8,9 and recently, has shown similar preliminary efficacy in higher risk MDS.10,11 Upregulation of another BCL2 family anti-apoptotic protein, MCL1, is seen in AML and MDS treated with VEN, and is a noted mechanism of VEN resistance.4,12-14 We previously revealed a selective MCL1 inhibitor with activity in AML patient samples dependent on MCL1 protein or resistant to BCL2 inhibition, including AML cells that arose from MDS.13 Taken together, targeting MDS cells via inhibition of BCL2 and/or MCL1 has appeal. We determined the sensitivity of MDS cells to inhibition of specific anti-apoptotic proteins, elucidated the characteristic determinants of response, and illustrated synergy with combined BCL2 and MCL1 inhibition both in vitro and in vivo in a murine xenograft model for MDS.
Patient and control samples
Experiments were conducted on primary MDS patient and normal (age range, 46-56 years [yr]; average, 50 yr) whole bone marrow (WBM) samples accessed from the Vanderbilt-Ingram Cancer Center Hematologic Malignancy Tumor Bank under a tissue collection protocol in accordance with the tenets of the Declaration of Helsinki and approved by the Vanderbilt University Medical Center Institutional Review Board. Normal CD34+ (age range, 26-44 yr; average, 31 yr) BM cells were purchased (Lonza and Hemacare).
Drug combination calculation of synergy
The effects of S63845 + VEN were calculated using the Zero Interaction Potency (ZIP) model, which compares observed and expected combination effects.15-17 The d synergy score predicts the likely interaction between two drugs (<-10: antagonistic; -10 to 10: additive; >10: synergistic).
Colony-forming unit assays
Cells (preparation described below) were plated in MethocultTM H4034 Optimum (STEMCELL Technologies) at a density of 0.05-2x104 cells per mL in the presence of different concentrations of VEN, S63845, or S63845 + VEN compounds. Colonies were counted and identified after 12-14 days by manual observation or by using the STEMvisionTM instrument and software analysis (STEMCELL Technologies). Counts were normalized per 1x104 MDS cells or per 500 normal CD34+ cells for graphical representation.
Cell preparation for colony-forming assays
Cryopreserved MDS whole bone marrow (WBM) or normal WBM and CD34+ cells were thawed and cultured in StemSpanTM (STEMCELL Technologies) + cytokines (10 ng/mL hIL3, 10 ng/mL hSCF, 5 ng/mL hIL6; Peprotech) overnight before plating cells in MethocultTM H4034 Optimum (STEMCELL Technologies). For MDS CD34+ cell plating, CD34+ cells were selected from WBM after thawing using the EasySepTM Human CD34 Positive Selction Kit II (STEM-CELL Technologies), and then cultured as described above. Fresh MDS BM cells were treated with 100 nM VEN, 100 nM S63845, or 100 nM of each compound for S63845 + VEN treatments in the same culture conditions as the growth inhibition assays. After 24 hours of treatment, cells were plated in MethocultTM H4034 Optimum at a density of 1-2x104 cells per mL. After colony counts were completed, the duplicate plates for each treatment group were washed with phosphate-buffered saline + 0.5% bovine serum albumin and pooled to obtain live cell number counts via trypan blue exclusion from the methocult assays on a subset of samples.
Generation of myelodysplastic syndrome patient-derived xenotransplants
All animal experiments were approved by the Institutional Animal Care and Use Committee of Yale University. Mouse breeding: MISh/hTRG6 mice with homozygous knockin replacement of the endogenous mouse Csf1, Il3, Csf2, Tpo, Il6 and Sirpa with their human counterparts were bred to MITRG6 mice to generate human cytokine homozygous and hSIRPA heterozygous mice (MISh/mTRG6, labeled MISTRG6 throughout the study). Mice were maintained on continuous treatment with enrofloxacin in the drinking water (0.27 mg/mL, Baytril, Bayer Healthcare). MISTRG/MISTRG6 mice will be available via MTA and requests should be sent to email@example.com.
Xenografting: newborn MISTRG6 mice (3 days of age) were X-ray irradiated (X-RAD 320 irradiator) with 2 × 150 cGy 4 hours apart. MDS patient BM CD34-selected cells were incubated with a murine anti-human CD3 antibody (clone Okt3, BioXCell) at 5 mg/100 mL for 10 minutes at room temperature prior to injection. Cells were injected intrahepatically in a volume of 20 mL with a 22-gauge Hamilton needle (Hamilton). Engraftment levels were assessed via BM aspiration at 12/ 16 weeks post-transplantation to assign treatment groups.
Data are shown as mean ± standard error of the mean (SEM) and analyzed using Prism 8 (GraphPad Software). Correlation analysis was determined using Spearman non-parametric correlation and statistical analysis was performed using an ANOVA (P<0.0001) followed by the student 2-tailed t-test. P values of <0.05 were considered to be statistically significant (n.s.: not significant, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001).
Myelodysplastic syndrome subtypes are differentially sensitive to BCL2 inhibition and indiscriminately sensitive to MCL1 inhibition
We successfully cultured 35 MDS patient samples and determined their in vitro sensitivity to BCL2, BCL-XL and MCL1 inhibitors. Using CellTiter-Glo, we generated dose response curves and determined the 50% growth inhibitory concentrations (GI50) for each inhibitor after 48 hours of exposure (Table 1). While few samples were sensitive to BCL-XL inhibition (A-1155463), we observed a range of sensitivities to BCL2 inhibition that positively correlated with blast count. Consistent with previous findings,1,4 lower blast count MDS (RS-SLD/MLD and MLD) exhibited less sensitivity to BCL2 inhibition than higher blast count MDS subtypes (e.g., MDS-EB1; -EB2) (P=0.004) (Online Supplementary Figure S1A). All subtypes were sensitive to the selective MCL1 inhibitor, S63845, with decreased sensitivity in the MDS-EB2 subtype. Samples were assessed using a targeted next generation sequencing (NGS) panel of 37 commonly mutated genes in myeloid diseases. As expected, we observed an increased number of SF3B1 mutations in the lower blast count MDS with ring sideroblasts (MDS-RS) samples.19 RAS-family mutants, particularly PTPN11-mutated AML have previously been shown to connote resistance to VEN.20-24 In this cohort, one of two PTPN11 mutant MDS samples, and two of two CBL mutant samples were completely resistant to BCL2 inhibition (MDS033, MDS005 and MDS031, respectively). Notably, the PTPN11 mutant sample that was not resistant to VEN (MDS026) had a low variant allele frequency (VAF) of 6% for this mutation whereas the other three RAS family mutants that demonstrated VEN-resistance all had VAF above 30%, which likely explains the lack of resistance to VEN in MDS026 (Online Supplementary Table S1). Otherwise, we did not observe any correlation between specific mutations and drug response in this cohort of 35 patient samples.
Dual inhibition of MCL1 and BCL2 is synergistic in all myelodysplastic syndrome samples resulting in increased apoptosis and loss of clonogenicity
In order to determine if co-inhibition of BCL2 and MCL1 is synergistic in MDS, we assessed the efficacy of three-fold dilutions of S63845 + VEN on cell viability and employed the ZIP synergy model to determine the synergistic potential.15 The combined treatment for all samples tested resulted in an average d synergy score >10, suggesting drug synergy (Figure 1A; Online Supplementary Table S2). In order to investigate if the reduced viability seen with dual inhibition of BCL2 and MCL1 led to apoptosis of MDS CD34+ progenitor cells, cells were stained with antihuman CD34, Annexin V, and propidium iodide (PI) 24 hours after treatment with sub-therapeutic doses of each inhibitor (Online Supplementary Figure S1B). Cells treated with S63845 + VEN displayed significant reductions in MDS progenitor cells compared to VEN-treated or S63845-treated cells when all MDS samples were analyzed together (Figure 1B). Similarly, a refined analysis of MDS subtypes revealed that combined treatment significantly reduced MDS progenitors for all subtypes compared to cells treated with S63845 alone. Compared to VEN alone, the dual treatment significantly reduced MDS progenitors in RS-SLD/MLD and MLD and trended toward statistical significance for EB1 and EB2 patient samples (Figure 1C). In order to determine the long-term effects of reduced viability and increased apoptosis on clonogenicity, colony-forming unit (CFU) assays were performed. In eight tested MDS patient WBM samples, dual treatment of 100 nM S63845 + 100 nM VEN reduced CFU-granulocyte magrophage (CFU-GM) formation by 50-100%, indicating loss of clonogenicity, even in CBL (MDS031) or PTPN11 (MDS033) mutant samples that were resistant to VEN monotherapy (82% and 50% reduction, respectively) (Figure 2A; Online Supplementary Figure 2B). This was further confirmed by trypan blue staining on cells collected from the plates of a subset of samples tested, which showed a reduction of total live cells (Online Supplementary Figure S4). In order to confirm dual inhibition of MCL1 and BCL2 reduced MDS HSPC, we verified this effect in CD34+ cells from four MDS patient samples. Similar to the effects on WBM, dual treatment with 100 nM S63845 + 100 nM VEN reduced CFU-GM formation in MDS HSPC by 42-100%, including a CBL (MDS005) and PTPN11 mutant sample that were resistant to VEN monotherapy (86% and 42%, respectively) (Figure 2A; Online Supplementary Figure S2D). Given the skewing toward CFU-GM colonies from the MDS samples that had been cryopreserved (Online Supplementary Figure S2B and D), we also verified the effects of S63845 + VEN treatment in freshly-obtained MDS patient samples, which corroborated a reduction, specifically, in CFU-GM colonies by 30-93% (Online Supplementary Figure S2E).
While 100 nM S63845 + 100 nM VEN was significantly more effective at reducing MDS WBM and HSPC colony formation, it was also toxic to normal hematopoiesis resulting in a 4-68% reduction in normal WBM CFU-GM colonies (n=8, mean 82% reduction in MDS-WBM-CFU-GM vs. n=4, mean 38% reduction in normal-WBM-CFU-GM, P=0.01) and a 38-69% reduction in normal CD34+ CFU-GM colonies (n=4, mean 80% reduction in MDS-CD34+-CFU-GM vs. n=7, mean 52% reduction in normal-CD34+-CFU-GM, P=0.03) (Figure 2A; Online Supplementary Figure S2A and C). In order to determine a therapeutic window that would not affect normal hematopoiesis, we tested a range of doses of BCL2 + MCL1 inhibition on normal CD34+ cells and found multiple dose combinations that had no significant impact on colony formation (Figure 2B). We reduced the original tested concentration by 50% and assessed dual treatment of 50 nM S63845 + 50 nM VEN, which exemplified the selective targeting of MDS compared to normal WBM cells (n=6, mean 50% reduction in MDS-WBM-CFU-GM vs. n=4, mean 0% reduction in normal-WBM-CFU-GM, P=0.0002) and HSPC (n=4, mean 71% reduction in MDS-CD34+-CFU-GM vs. n=2, mean 0% reduction in normal-CD34+-CFU-GM, P=0.02) (Figure 2C; Online Supplementary Figure S3A to D). Markedly, all MDS WBM and CD34+ samples displayed reduced CFU-GM colony formation using either high (100 nM S63835 + 100 nM VEN) or low (50 nM S63845 + 50 nM VEN) combination therapy, even VEN-monotherapy-resistant (GI50 >5 mM) samples (Online Supplementary Table S3).
Combined MCL1 and BCL2 inhibition reduces human myelodysplastic syndrome cell engrafment in MISTRG6 mice
We further validated the efficacy of S63845 + VEN combination treatment using in vivo MDS patient-derived xenotransplants (PDX). We have previously shown successful establishment of MDS PDX with faithful clonal representation in cytokine humanized ‘MISTRG’ mice. MISTRG mice carry humanized alleles for M-CSFh/h, IL-3/GM-CSFh/h, SIRPah/m, and TPOh/h, on the RAG2-/- γc-/- background, and are viable, healthy and fertile. Gene-humanizations via knockin significantly improve the engraftment, differentiation and maintenance of human cells from MDS and other hematologic malignancies.25-27 As MDS is an inflammatory disorder with elevated IL-6 levels and is highly dependent on its microenvironment,28,29 we made use of a next-generation version of MISTRG with humanization via targeted insertion of human interleukin-6 (IL-6), in short, ‘MISTRG6’. MISTRG6 have been shown recently to efficiently engraft healthy human huCD34+ and myeloma cells.26,30 Before testing the effects of combined BCL2 and MCL1 inhibition experiments in MDS PDX, we performed toxicity evaluations in MISTRG6 mice. Mice were treated with vehicle, VEN (15 mg/kg; 5 days on, 2 days off), or a combination of VEN (15 mg/kg; 5 days on, 2 days off) and S63845 (12.5 mg/kg; 2 days on, 5 days off) for a total of 2 weeks. Measurements were taken prior to starting treatment (Pre), 1 week, and 2 weeks after treatment to determine weight, red blood cell count, hemoglobin, white blood cell count, and platelet count. No detrimental effects were seen on the mouse hematopoietic cells in MISTRG6 mice (Online Supplementary Figure S5). Next, we confirmed engraftment of selected MDS patient samples from Table 1 that had sufficient cells to perform in vivo studies. All three samples engrafted in the MISTRG6 mice, at varying levels in the bone marrow (BM) (Figure 3A and B), and were largely comprised of the myeloid lineage (Figure 3C). In order to maximize the information we could obtain with this limited resource, we used these test engraftment mice to perform a pilot study comparing vehicle treatment to combined BCL2 + MCL1 inhibition. This preliminary experiment suggested that dual treatment would decrease MDS engraftment in the BM (Online Supplementary Figure S6).
In vitro assays showed patient sample MDS019 was resistant to VEN treatment alone (Table 1; GI50 =1.6 mM), but responded to dual BCL2+MCL1 inhibition (Online Supplementary Table S2; average ZIP d synergy score =21.6). Since MDS019 also led to the most consistent engraftment (Figure 3A and B), we tested this sample to determine if combined BCL2 + MCL1 inhibition could also overcome VEN resistance in vivo. Remaining cells from MDS019 (MDS-EB1 subtype) were allowed to engraft in MISTRG6 mice for 16 weeks before beginning treatment with vehicle, VEN (15 mg/kg; 5 days on, 2 days off), or a combination of VEN (15 mg/kg; 5 days on, 2 days off) and S63845 (12.5 mg/kg; 2 days on, 5 days off) for a total of 4 weeks (n=4 per treatment group). The percent or total number of human CD45+ (huCD45+) cells in the BM were compared between pre- and post-treatment for each treatment group (Figure 4A and B, respectively). VEN single treatment reduced the number of huCD45 cells in the BM but displayed a marked increase in the percent of huCD45 cells similar to mice treated with vehicle. Conversely, both of these analyses demonstrated a striking reduction in percent and number of huCD45+ cells in the BM after combined BCL2+MCL1 inhibition, with almost a complete loss of huCD45+ cells. Furthermore, after the completion of the treatment, measurement of the percent of huCD45+/lin-/huCD38-/huCD34+ HSPC in the BM (Figure 4C) indicated this combined treatment, but not VEN treatment alone, was successful at specifically reducing the MDS-HSPC that are known to drive the propagation of MDS. This finding was further exemplified by reduced CD34+ staining by immunohistochemistry in the bone marrow of mice treated with dual BCL2 + MCL1 inhibition (Figure 4D). Notably, these results are consistent with the outcome of the in vitro proliferation assays for MDS019 patient cells.
The study of MDS HSPC in vitro is challenging. MDS cell lines are limited, contrived and not representative of MDS, particularly lower risk MDS, so patient samples are required.31-33 Ex vivo study of MDS patient samples is also limited by sample heterogeneity, availability, and the practical consideration of how few cells are available in the setting of a disease characterized by marrow failure. Nonetheless, we have performed a comprehensive analysis on a cohort of MDS patient samples to investigate the value of targeting anti-apoptotic proteins for the treatment of MDS. Our data validated previous findings that higher blast count MDS subtypes (EB1 and EB2) are more sensitive to VEN monotherapy than low blast count subtypes.1,4 We illustrated that mutations associated with VEN resistance in AML may be similarly relevant in MDS, and also discovered, importantly, that MCL1 inhibtion may be effective for all MDS subtypes. Moreover, drug synergy from combining BCL2 and MCL1 inhibitors can be achieved across all subtypes and mutational profiles of MDS in this model, even in the presence of RAS family mutations that are believed to confer resistance to VEN monotherapy (e.g., CBL and PTPN11 in this cohort).20-24
The in vivo reduction of MDS progenitor cells in the BM from MDS PDX is the first successful test of selective anti-apoptotic inhibitors in an in vivo MDS PDX model. Prior patientderived MDS xenograft models have demonstrated inefficient engraftment potential in multiple murine backgrounds and often do not recapitulate the disease characteristics.32,34-42 Other studies have shown mixed results when attempting to improve MDS cell engraftment into mice with co-injection of normal or MDS patient-derived mesenchymal stromal cells.40,42 The MISTRG mice overcame the obstacles presented by other models by expressing human cytokines in place of their murine counterparts at the endogenous locus to provide physiological expression of these human cytokines and MISTRG6 mice further provide the otherwise non-crossreactive critical inflammatory cytokine, IL-6, which is over-expressed in mesenchymal stromal cells in MDS and other hematologic malignancies.25 Building on this model allowed us to demonstrate successful engraftment of MDS patient cells in MISTRG6 mice for the first time, and to test the effects of anti-apoptotic inhibitors in vivo. Xenografting of human MDS is improved with the MISTRG6 model, yet a finite number of patient samples with robust cellular engraftment hindered the number of transplants. Nonetheless, these MDS PDX experiments edify findings from our ex vivo studies to illustrate the selective utility of BCL2 inhibitor monotherapy in MDS and provide promise for the use of combined BCL2 + MCL1 inhibition for the treatment of all MDS subtypes, particularly in patients who may be resistant to VEN monotherapy.
BCL2 inhibition has changed the standard of care in myeloid malignancies; refining the design of clinical trials testing BCL2 and MCL1 inhibitors in MDS and the precision of patient selection for therapy is a top therapeutic priority. While combination of BCL2 and MCL1 inhibition may have a dose-dependent impact on normal hematopoiesis, experimental evidence seems to indicate that toxicity on normal CD34+ cells is limited at clinically meaningful doses in vivo13 and in vitro,43 as we have also shown here. The data presented here suggest dual BCL2 + MCL1 inhibition should be considered for the treatment of all MDS subtypes regardless of mutational status, even those with mutations that may be less likely to respond to VEN monotherapy (e.g., TP53 or RAS family mutations),20-24 and carefully designed clinical trials with flexible dosing schedules amenable to MDS are warranted.
- Received January 10, 2022
- Accepted August 11, 2022
MRS has served on consultancy/advisory board/data safety monitoring committees for Abbvie, BMS, CTI, Forma, Geron, Karyopharm, Novartis, Ryvu, Sierra Oncology, Takeda, Taiho TG Therapeutics; has equity in Karyopharm; and his institution has received research funding from ALX Oncology, Incyte, Takeda, and TG Therapeutics. SH serves on the advisory board of FORMA Therapeutics.
MAF, YS, and RG designed and performed experiments. MPA, and MTV performed experiments. MAF, YS, RG, MPA, MTV, MAC, BNS, TPS, SH, and MRS analyzed data. MAF and MRS performed statistical analysis and wrote the manuscript. SH and MRS designed and supervised the study. All authors reviewed and edited drafts of the manuscript and approved the final version of the manuscript.
Not applicable as data generated in this study have been included in the published article.
This work was generously supported by the Edward P. Evans Foundation (2017-11), Beverly and George Rawlings Directorship, the Biff Ruttenberg Foundation, the Adventure Alle Fund, and the Leukemia and Lymphoma Society (2321-19) for which, MRS is a Clinical Scholar. MRS was supported by the NCI (R01CA262287-01). SH was supported by the NIH/NIDDK (R01DK102792), the Frederick A. Deluca Foundation, the Edward P. Evans Foundation, and the Department of Defense. YS was supported by a pilot grant from the Yale Cooperative Center of Excellence in Hematology (YCCEH) and the National Natural Science Foundation of China (General Program No. 82170137). BNS was supported by the NCI (5K12CA090625). This study was in part supported by the Animal Modeling Core of the Yale Cooperative Center of Excellence in Hematology (NIDDK U54DK106857). Patient samples were garnered from the Vanderbilt-Ingram Cancer Center (VICC) Hematologic Malignancy Tumor Bank. The Vanderbilt-Ingram Cancer Center grant (NCI P30 CA068485-19) supported this work. The REDCap database tool is supported by grant UL1 TR000445 from NCATS/NIH. Flow Cytometry experiments were performed in the VUMC Flow Cytometry Shared Resource. The VUMC Flow Cytometry Shared Resource is supported by the Vanderbilt Ingram Cancer Center (P30 CA68485) and the Vanderbilt Digestive Disease Research Center (DK058404).
For the in vivo studies, we thank Yale Pathology Tissue Services, especially A. Brooks for research histology services, Yale Animal Resources Center, especially P. Ranney, J. Fonck for animal care, and Yale Flow Cytometry Core, especially Lesley Divine, Diane Trotta and Chao Wang for flow cytometry analysis.
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