Enhanced p53 Activation By Dual Inhibition of MDM2 and XPO1 Disrupts MYC Transcriptional Program and Restores Sensitivity to BCL-2 Inhibition in Ven/HMA Resistant AML
Authors: Nishida Yuki , Ishizawa Jo , Ayoub Edward , Patsilevas Tallie , Ostermann Lauren , Montoya Rafael Heinz , Muftuoglu Muharrem , Ruvolo Vivian , Mak Po Yee , Tao Wenjing , Carter Bing Z , Kojima Kensuke , Daver Naval , Lesegretain Arnaud , Seki Takahiko , Shacham Sharon , Konopleva Marina , Andreeff Michael 
Published: November 2021

Abstract
The BCL-2 inhibitor venetoclax (ven) in combination with hypomethylating agents (HMA) has revolutionized acute myeloid leukemia (AML) therapy. However, the majority of patients with AML who received ven/HMA eventually relapse and cures are still elusive. We previously generated ven-resistant MV4;11 (MV4;11 VR) cells and found them to express elevated c-Myc protein levels. The dual inhibition of MDM2 and XPO1 significantly increased nuclear p53 protein and dramatically upregulated p53 target genes. On the other hand, dual inhibition of MDM2 and XPO1 profoundly decreased c-Myc protein levels in a p53-dependent manner, resulting in the profound downregulation of MYC transcriptional program. Clinical grade MDM2 and XPO1 inhibitors milademetan (mil) and selinexor (sel) significantly reduced leukemia burden and prolonged survival in the xenograft model injected with MV4;11 VR cells. However, the treatment effect did not result in very long relapse free responses. To investigate potential resistance mechanisms to dual inhibition of MDM2 and XPO1, we utilized our previously reported (Muftuoglu, ASH 2020) multiparameter flow cytometry to assess multiple stress responses pathways including p21, ATF4, PARP, LC3B, Ki-67, active caspase-3 and amine-reactive viability dye at the single cell level in ven-resistant AML cells. We found that residual cells, after combined MDM2 and XPO1 inhibition, expressed high levels of p21, ATF4 and LC3B and low Ki-67 levels, suggesting that the resistant population is in a cell kinetic quiescent state with activation of autophagy and ER stress. Interestingly, ven-resistant MV4;11 cells with in vivo acquired resistance to dual inhibition of MDM2 and XPO1 demonstrated elevated protein levels of respiratory chain complex proteins (NDUFB8, MTCO1, UQCRC2 and ATP5A), suggesting increased OXPHOS dependency. Intriguingly, these MV4;11VR cells with subsequently acquired resistance to MDM2 and XPO1 inhibition in vivo have restored sensitivity to ven. We previously reported that the combination of MDM2 and BCL-2 inhibitors induces synergistic apoptosis through the elimination of dormant p21 high residual AML cell (Pan, Cancer Cell 2017). In a clinical trial of Idasanutlin + Venetoclax we observed over 45% response rates in relapsed/refractory AML patients (Daver, ASH 2020). Therefore, we hypothesized that combining MDM2/XPO1 inhibition with BCL-2 inhibition further induces cell killing in ven/HMA resistant AML cells.
To address this, we treated ven-resistant AML cells with triple combination of mil, sel and ven, resulting in a profound cytoreduction in vitro compared with other single and doublet treatments. Next, we treated NSG mice injected with PDX AML cells with FLT3-ITD, GATA2 and NRAS mutations obtained from a patient who relapsed after ven/decitabine treatment. The doublet combinations especially of mil + ven reduced circulating blasts and significantly prolonged survival. Of note, no mice have died at day 180 after treatment in the group receiving triple combination of mil, ven and sel, with profound and sustained cytoreduction (more than 2 log 10 difference) and significantly prolonged survival compared with any other treatment (Fig. A, B). The triple combination was well-tolerated and did not decrease mouse CD45+ cells, platelets and hemoglobin.
In conclusion, the concomitant inhibition of MDM2, XPO1 and BCL-2 was feasible and exerted dramatic and sustained anti-leukemia activity in ven/HMA resistant AML cell in vitro and in vivo. Milademetan (RAIN-32) is currently being developed by Rain Therapeutics.
Figure 1 Figure 1.

Disclosures
Carter: Syndax: Research Funding; Ascentage: Research Funding. Daver: Pfizer: Consultancy, Research Funding; Jazz Pharmaceuticals: Consultancy, Other: Data Monitoring Committee member; Novartis: Consultancy; Gilead Sciences, Inc.: Consultancy, Research Funding; Daiichi Sankyo: Consultancy, Research Funding; Bristol Myers Squibb: Consultancy, Research Funding; ImmunoGen: Consultancy, Research Funding; Trillium: Consultancy, Research Funding; Glycomimetics: Research Funding; Sevier: Consultancy, Research Funding; Astellas: Consultancy, Research Funding; Genentech: Consultancy, Research Funding; Abbvie: Consultancy, Research Funding; Novimmune: Research Funding; Hanmi: Research Funding; Amgen: Consultancy, Research Funding; Trovagene: Consultancy, Research Funding; FATE Therapeutics: Research Funding; Dava Oncology (Arog): Consultancy; Celgene: Consultancy; Syndax: Consultancy; Shattuck Labs: Consultancy; Agios: Consultancy; Kite Pharmaceuticals: Consultancy; SOBI: Consultancy; STAR Therapeutics: Consultancy; Karyopharm: Research Funding; Newave: Research Funding. Lesegretain: Daiichi-Sankyo Inc.: Current Employment. Seki: Daiichi-Sankyo Inc.: Current Employment. Shacham: Karyopharm: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties: (8999996, 9079865, 9714226, PCT/US12/048319, and I574957) on hydrazide containing nuclear transport modulators and uses, and pending patents PCT/US12/048319, 499/2012, PI20102724, and 2012000928) . Konopleva: Ascentage: Other: grant support, Research Funding; Reata Pharmaceuticals: Current holder of stock options in a privately-held company, Patents & Royalties: intellectual property rights; Eli Lilly: Patents & Royalties: intellectual property rights, Research Funding; Novartis: Other: research funding pending, Patents & Royalties: intellectual property rights; AstraZeneca: Other: grant support, Research Funding; Rafael Pharmaceuticals: Other: grant support, Research Funding; F. Hoffmann-La Roche: Consultancy, Honoraria, Other: grant support; AbbVie: Consultancy, Honoraria, Other: Grant Support, Research Funding; Genentech: Consultancy, Honoraria, Other: grant support, Research Funding; Forty Seven: Other: grant support, Research Funding; Cellectis: Other: grant support; Calithera: Other: grant support, Research Funding; Agios: Other: grant support, Research Funding; KisoJi: Research Funding; Ablynx: Other: grant support, Research Funding; Stemline Therapeutics: Research Funding; Sanofi: Other: grant support, Research Funding. Andreeff: Medicxi: Consultancy; Glycomimetics: Consultancy; AstraZeneca: Research Funding; Syndax: Consultancy; Amgen: Research Funding; Oxford Biomedica UK: Research Funding; Karyopharm: Research Funding; Daiichi-Sankyo: Consultancy, Research Funding; Breast Cancer Research Foundation: Research Funding; Novartis, Cancer UK; Leukemia & Lymphoma Society (LLS), German Research Council; NCI-RDCRN (Rare Disease Clin Network), CLL Foundation; Novartis: Membership on an entity's Board of Directors or advisory committees; Senti-Bio: Consultancy; Reata, Aptose, Eutropics, SentiBio; Chimerix, Oncolyze: Current holder of individual stocks in a privately-held company; ONO Pharmaceuticals: Research Funding; Aptose: Consultancy.