Targeting Autophagy Kinase ULK1 Can Reverse Bcl2 Inhibitor (ABT-199) Induced Autophagy to Overcome Acquired Resistance in Acute Myeloid Leukemia

Introduction

Anti-apoptotic Bcl2 family members mediate resistance to therapies in acute myeloid leukemia (AML)¹. The small molecule Bcl2 inhibitor ABT-199 (venetoclax) promotes mitochondria driven intrinsic apoptosis, and in combination with hypomethylating agents or chemotherapy, has been highly promising in the clinic as treatment of AML^2-4. The response rate to ABT-199 is very impressive, but acquired resistance is a major problem. Compensatory upregulation of Mcl1 is an important mechanism of such acquired resistance to mitochondrial apoptosis⁵. Autophagy is vital for mitochondrial health, mediates resistance to apoptosis and is induced by Bcl2 inhibition⁶. We performed mechanistic studies to address our hypothesis that disabling autophagy by targeting the apical autophagy kinase ULK1 can reverse resistance to ABT-199.

Methods

ULK1 was genetically modified in OCIAML3 (human AML cell line), by shRNA knockdown (KD) or CRISPR-Cas9 knockout (KO). In addition, AML cell lines (including ABT-199 resistant) and patient samples were treated with ABT-199 and ULK1 inhibitor SBI-0206965⁷. Combination index (CI) for drug synergy was calculated based on Chou-Talalay method⁸. Drug-treated or genetically manipulated cells were profiled by reverse phase protein array (RPPA), mass cytometry (CyTOF) and gene expression profiling (GEP). Autophagy was detected by LC3 quantification by western blot (WB) and flow cytometry, and monodansylcadaverine assay. Mitochondrial functions were analyzed by Seahorse Cell Mito Stress test, and MTG, TMRE and ROS assays (flow cytometry). For in vivo studies ULK1 KO and corresponding control cells were injected in NSG mice and monitored by bioluminescent imaging (BLI) and quantification of human CD45 cells.

Results

ABT-199 induced autophagy in OCIAML3 (increase by 175±27%, p=0.01 - LC3 flow; 4X increase in LC3 II/I ratio - WB). Apoptosis induction by ABT-199 was enhanced by ULK1 KD (36±1.9% over control, p<0.01) or KO (77±1.3%, p<0.01). ULK1 inhibitor SBI-0206965 demonstrated high synergy with ABT-199 at inducing apoptosis in OCIAML3 (CI = 0.51, p<0.001) and MOLM13 (CI = 0.24, p<0.001). The combination was also effective in eliminating bulk and CD34+ stem/progenitor cells in primary AML samples (CI - Bulk: 0.69; CD34: 0.74; p<0.05) (Fig 1).

Mcl1 was significantly downregulated by ULK1 inhibitor alone and in combination with ABT-199. ULK1 inhibition lowered Mcl1 transcription, as measured by qRT-PCR: 43±0.03% with SBI-0206965 and 63±0.3% in KO cells (both p<0.01). SBI-0206965 inhibited Mcl1 transcription regulators Stat3 and Erk1/2 and enhanced DNA damage in combination with ABT-199 (WB) (Fig 2).

Since ABT-199 modulates mitochondrial function, we examined the effect of inhibiting ULK1 in this context. By Seahorse assay, the combination decreased basal OCR and ATP production by 62 and 58% respectively, p<0.01. This was accompanied by an increase in membrane depolarization (TMRE change - OCIAML3: 62%, MOLM13: 82%; p<0.01) and mitochondrial ROS generation (62% increase, p=0.01) compared to control cells (Fig 3). Interestingly, ULK1 inhibition increased mitochondrial mass (30%, p=0.012) by MTG assay, which may be due to impaired mitophagy. Thus, the combination impairs mitochondrial metabolism and function, which results in the observed increase in ROS that may account for the observed DNA damage and apoptosis. CD44/44v is critical for mitigating ROS through reduced glutathione (GSH)⁹, and ULK1 inhibition lowered CD44/44v transcription (qRT-PCR) and intracellular GSH in AML cells.

Corroborating our earlier data, the ABT-199 resistant cells (OCIAML2R & MOLM13R) show enhanced autophagy as compared to parental cells (OCIAML2: 83%, MOLM13: 35% increase; p=0.001 & 0.009). SBI-0206965 reversed ABT-199 induced autophagy and restored ABT-199 sensitivity in these cells (Fig 4).

In a pilot in vivo experiment control and ULK1 KO cells were injected in NSG mice and leukemia engraftment was markedly delayed in the ULK1 KO group (Fig 5). The therapeutic combination study is ongoing.

Conclusion

Results indicate concomitant targeting of autophagy by ULK1 inhibition and Bcl2 inhibition by ABT-199 can overcome acquired resistance to ABT-199. Hence, with the emergence of Bcl2 inhibitors in frontline therapy for AML and efforts at developing ULK1 inhibitors, this study informs the development of novel apoptosis/autophagy targeting approaches to improve AML therapy.

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