Combination of the Kdm-Family Inhibitor, Jib-04, Improves Response to Acute Myeloid Leukemia Cells to Venetoclax

Background: Acute Myeloid Leukemia (AML) is a heterogeneous hematological malignancy with a 5-year survival rate of about 30% in adults with AML. While over the prior several decades, the standard of care treatment for AML changed very little, a number of novel targeted therapies have emerged in recent years. Promising results from clinical trials with the BCL2-specific inhibitor, Venetoclax, in combination with hypomethylating agents (HMA) or low-dose cytarabine for AML treatment have generated enthusiasm in the field. However, data from a phase II trial indicated a 30% failure rate after Venetoclax+HMA treatment of newly diagnosed patients, suggesting additional options are needed. Thus, we sought to explore alternative drug combinations that may enhance the efficacy of Venetoclax. JIB-04 is a potent small molecule inhibitor of multiple members of the histone lysine demethylase (KDM) 4, 5 and 6 families with KDM5A being the most sensitive. Expression levels of histone lysine-specific demethylases are higher in AML compared to normal bone marrow, suggesting they are potential targets for therapeutic intervention. Pharmacological inhibition or genetic knock down of KDM 4, 5 and 6 family members leads to reduced proliferation, cell cycle arrest, and increased apoptosis of AML cells of various subtypes. These preliminary findings compelled us to explore the anti-leukemic activity of JIB-04 alone and in combination with Venetoclax.

Methods: We treated AML cell lines, patient AML blasts, and CD34+ cells from healthy donors with JIB-04, Venetoclax, or the combination for 72 hours in vitro. Apoptosis was quantified by AnnexinV/propidium iodide (PI) staining. Cell proliferation of AML cell lines treated with JIB-04 was evaluated using MTT assay and transcriptome profiling was done by RNA-Seq. NSG mice were injected with MOLM-13 cells and treated with JIB-04, Venetoclax or the combination for two weeks. For the knock-down (KD) of DDIT4 MOLM-13 cells were transduced with a shRNA and cells were treated with Venetoclax for 72h and analyzed for apoptosis.

Results: Treatment of AML cell lines with low nanomolar doses (IC50 range from 8-60nM) of JIB-04 resulted in inhibition of proliferation and induction of apoptosis. In all AML cell lines tested, including AML cell lines with relative resistance to Venetoclax, the combination of JIB-04 with Venetoclax showed a strong synergistic effect. Moreover, patient AML blasts also showed a synergistic response to the drug combination compared to single-drug treatments (Figure 1a). In contrast, viability of normal CD34+ hematopoietic cells was not affected by JIB-04 treatment even up to a dose of 100nM that leads to apoptosis in AML cells, indicating a broad therapeutic window. The combination treatment of NSG mice injected with MOLM-13 cells resulted in reduced engraftment of huCD45+ cells compared to the single treatments in the blood, bone marrow and spleen. RNA sequencing of AML-OCI3 and MOLM-13 cells treated with JIB-04 revealed regulation of genes related to the mTOR pathway, stress response or amino acid metabolism, indicating possible mechanisms affecting proliferation and apoptosis. DDIT4 (REDD1), a regulator of the mTOR pathway, was downregulated after JIB-04 treatment in both cell lines. KD of DDIT4 in MOLM-13 cells resulted in increased sensitivity to Venetoclax (Figure 1b).

Summary/Conclusion: While the epigenetic inhibitor JIB-04 alone shows modest anti-leukemic effect, this study highlights the potential therapeutic benefit of combining JIB-04 and Venetoclax in AML. The analysis of transcriptomic changes after JIB-04 treatment identified the mTOR pathway regulator DDIT4 as a likely mediator of the sensitization of AML cells to Venetoclax. High DDIT4 expression is associated with a worse outcome in AML making it an attractive therapeutic target for AML, especially in combination with Venetoclax. Our result would suggest this drug combination as a possible treatment option for AML patients.

Rau:AbbVie Pharmaceuticals: Other: Spouse is employee and stock holder; Jazz Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Servier Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees.