Identifying and Overcoming Therapeutic Resistance in High-Risk Acute Lymphoblastic Leukaemia

Abstract

Background: Acute lymphoblastic leukaemia (ALL) is the most common childhood cancer and early disease eradication is critically important for long-term cure. ALL remains a leading cause of cancer death in children and young adults because of treatment toxicity and relapsed disease. Increased delineation of the key biological drivers in ALL as well as therapeutic response will present new opportunities to rationally combine existing and novel agents to improve outcomes whilst minimising toxicities.

Blocks in apoptosis are now widely recognised as a hallmark of ALL but also a mechanism of resistance to standard chemotherapeutic agents. Co-targeting aberrant cell survival pathways, using novel combinations of BH3-mimetics, are an emerging therapeutic option. My thesis centres on translational and mechanistic studies of combining venetoclax (BCL-2 inhibitor) and S63845 (MCL-1 inhibitor) in high-risk (HR) ALL subtypes.

Aim: To inform the clinical utility of BH3-mimetic combinations in HR-ALL by identifying synergistic combinations with each other as well as standard and targeted agents in vitro, evaluating the efficacy and tolerability of combination venetoclax (BCL-2 inhibitor) and S63845 (MCL-1 inhibitor) in vivo, and investigating mechanisms of therapeutic resistance.

Methods: BH3-mimetics were combined with each other as well as dexamethasone and targeted tyrosine kinase inhibitors (TKI) to identify the most potent combinations across kinase-activated ALL cell lines and patient derived xenografts (PDX) in vitro and in vivo. The tolerability of combinations venetoclax and S63845 was detailed in NOD-SCID-IL7R (NSG) mice as well as healthy donor blood ex vivo. Loss-of-function mutations that confer resistance to TKIs and BH3-mimetics in a Ph+ALL cell line were identified using an unbiased genome-wide CRISPR-Cas9 loss-of-function screen. Differences in cell signalling and survival pathways following treatment with TKI and BH3-mimetics in vivo in PDX models of Ph-like ALL were identified using mass cytometry.

Results: BCL-2 and MCL-1 protein expression remained high in kinase-activated B-ALL cell lines treated with targeted TKI despite up-regulation of BIM pro-apoptotic protein expression. Co-inhibition of BCL-2 and MCL-1, with combination venetoclax and S63845, induced synergistic killing in vitro and was comparable or superior to steroid or TKI combined with each BH3-mimetic, across a range of kinase-activated B-ALL cell lines and PDXs, including the Ph+ and Ph-like subtypes. The combination also had potent anti-leukaemia activity in vivo, which was demonstrated by rapid cytoreduction, but also acute tumour lysis syndrome (ATLS) in some PDX models of Ph-like B-ALL. Combining venetoclax and S63845 appeared tolerable, however, histologic evidence of haematopoietic toxicity was observed, at higher doses, in NSG mice, and synergistic cytotoxicity was observed in lymphocytes of healthy donor blood. Loss of function mutations in NOXA and BAX were identified from the CRISPR screen as important causes of resistance to dasatinib and venetoclax in Ph+ALL. Lastly, CyTOF demonstrated distinct single cell variability in response to TKI or BH3-mimetic treatment of Ph-like ALL, including differences in surface marker expression (CD38, CD179a, and CD34), and cell signalling pathways (pSTAT5).

Conclusion: Co-inhibition of BCL-2 and MCL-1 induces synergistic killing in vitro and rapid cytoreduction in vivo in a range of HR B-ALL models, including the Ph+ and Ph-like subtypes. The combination of BCL-2 and MCL-1 inhibition was comparable or superior to steroid or TKI combined with each BH3-mimetic. Although this combination of BH3-mimetics was tolerable in vivo at lower doses, histologic evidence of haematopoietic toxicity and tumour lysis syndrome was observed in NSG mice and PDX models, respectively. The expression level of BCL-2 family anti-apoptotic genes (BCL-2 and MCL-1), BH3-only class of pro-apoptotic genes (NOXA and BAX), surface markers (CD38, CD179a, and CD34) and cell signalling pathways (pSTAT5), predicted treatment resistance. Co-targeting BCL-2 and MCL-1 warrants evaluation in clinical trials that incorporate supportive care including infection prophylaxis and tumour lysis precautions. The findings from this research may be exploited for future studies to test novel combinations of drugs, on the basis of their ability to act on non-overlapping mechanisms, which are likely to result in synergistic anti-leukaemia efficacy.