Identification of Potent BH3-Mimetic Combinations Targeting Pro-Survival Pathways in Human B-Cell Acute Lymphoblastic Leukemia

Background

Precursor-B acute lymphoblastic leukemia (B-ALL) is an aggressive hematological malignancy. Relapsed disease has a poor prognosis, despite improved outcomes with tyrosine kinase inhibitors for Ph+ cases and immunotherapeutic approaches, such as blinotumomab and CAR-T cells. Targeting cell survival with novel small molecule BH3-mimetic inhibitors of BCL-2 (e.g. Souers et al Nat Med 2013, Roberts et al, NEJM 2016 and Casara et al, Oncotarget 2018), BCL-X_L (Lessene et al, Nat Chem Biol, 2013) or MCL1 (Kotschy et al, Nature 2016) is an emerging therapeutic option. BCL-2 is reported to have a pro-survival role in BCR-ABL1, JAK2 fusion, ETV6-RUNX1 and MLL-r driven ALL (Brown et al., Journal Biological Chemistry 2017). BH3-mimetics targeting BCL-2 and BCL-X_L has efficacy in paediatric ALL xenografts (Khaw et al., Blood 2016), while ruxolitinib combined with ABT-737 is synergistic in JAK2-mutant pre-B-ALL (Waibel et al., Cell Reports 2013). We now report that combined targeting of BCL-2 and MCL1 has broad pre-clinical efficacy in adult B-ALL samples with Ph+, Ph- and Ph-like characteristics.

Methods

S55746 and S63845 were obtained from Servier/Novartis, A1331852 from Guillaume Lessene (WEHI), venetoclax, daunorubicin, dexamethasone (DXM) and tyrosine kinase inhibitors (TKIs) from Selleckchem. Bliss synergy scores were determined using a checkerboard approach to evaluate combinations (previously described Bliss, Ann Appl Biol 1939). Primary ALL cells were obtained from 14 patients (4 Ph+ and 10 Ph-) providing informed consent. Ex vivo cell viability (sytox blue exclusion) at 48h was determined over a 5-log dilution range (1nM-10uM) using drugs alone or in equimolar combinations. For in vivo studies, adult B-ALL patient derived xenografts were performed in NSG; NOD.Cg-Prkdc^scid Il2rg^tm1Wjl/SzJ mice.

Results

Dual BH3-mimetic targeting of BCL-2 and MCL1 was strongly synergistic (Bliss sum >1000) in SUPB15 (Ph+ BCR-ABL1), BV173 (lymphoid blast crisis BCR-ABL1), MUTZ5 (Ph-like) and MHHCALL4 (Ph-like) B-ALL cell lines. This was more effective than single BH3-mimetic combinations with DXM or TKIs (dasatinib or ruxolitinib) (Fig. A, B). In B-ALL patient samples, combined BCL-2 and MCL1 targeting lowered the LC₅₀ by 10-1000 fold (to LC₅₀<10nM) in 4/4 Ph+ ALL cases and 8/10 Ph- cases. Similarly, combined MCL1 and BCL-X_L targeting demonstrated synergy in 3/4 Ph+ cases and 7/10 Ph- cases (to LC₅₀<10nM), confirming remarkable anti-leukemic activity compared to BH3-mimetics alone or chemotherapy (daunorubicin) (Fig. C). BH3-mimetic combination therapy (S55746/S63845) compared favourably in Ph+ ALL cases to S55746 (figure D) or S63845 (Figure E) in combination with dasatinib. Preliminary data using patient-derived xenografts in NSG mice revealed in vivo efficacy of combined S55746 and S63845 therapy against 3 adult B-ALL cases (1 Ph+ and 2 Ph-). Reduction of established ALL in the bone marrow was observed in mice receiving combined S55746/S63845 after one week of treatment (p=<0.05) (Fig. F-H).

Conclusions

Dual BH3-mimetic targeting of BCL-2 and MCL1 induces synergistic killing of human B-ALL cell lines and primary ALL samples in vitro and rapid cytoreduction in vivo. Simultaneous inhibition of BCL-2 and MCL1 represents a novel and effective approach for targeting Ph+, Ph- and Ph-like B-ALL without need for additional DNA-damaging chemotherapy or kinase inhibition. Our results support the translational investigation of dual BH3-mimetic targeting of BCL-2 and MCL1 in the clinic.

Figure legend: BLISS synergy scores for A. Ph+ and B. Ph-like ALL cell lines for drug combinations targeting BCL-2, MCL1, BCR-ABL, JAK1/2 and DXM. C. LC₅₀ activity in primary ALL after 48hr of treatment with BH3 mimetics and combinations targeting BCL-2, MCL1, BCL-X_L, compared to daunorubicin (LC50< 10nM red; ~ 100nM yellow; >1uM green). D. Comparison of BH3-mimetics targeting D.BCL-2 or E. MCL1 in combination with dasatinib in Ph+ vs Ph- primary B-ALL samples. Activity expressed as LC₅₀ activity after 48h, with median values shown. Irradiated NSG mice were transplanted with 10⁶ primary B-ALL cells. Engraftment of F. Ph+ and G-H. Ph- B-ALL cells was confirmed at 10 weeks by detection of hCD45 in PB. Mice were then treated with i) vehicle (d1-5), ii) S55746 100mg/kg days 1-5 by gavage, iii) S63845 25 mg/kg IV on days 2 and 4 or iv) S55746+S63845. Mice were euthanized on day 8 and hCD45+ from flushed femurs quantified.

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