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2770 The Menin Inhibitor Ziftomenib (KO-539) Synergizes with Agents Targeting Chromatin Regulation or Apoptosis and Sensitizes AML with MLL Rearrangement or NPM1 Mutation to Venetoclax

Program: Oral and Poster Abstracts
Session: 616. Acute Myeloid Leukemias: Investigational Therapies, Excluding Transplantation and Cellular Immunotherapies: Poster II
Hematology Disease Topics & Pathways:
Research, apoptosis, Translational Research, Combination therapy, Therapies, Biological Processes, molecular biology
Sunday, December 11, 2022, 6:00 PM-8:00 PM

Johanna Rausch, MD1*, Margarita M. Dzama, PhD1*, Nadezda Dolgikh, PhD1*, Hanna Stiller1*, Stephan Bohl, MD2*, Catharina Lahrmann1*, Kerstin Kunz1*, Linda Kessler, BA3*, Hakim Echchannaoui, PhD1*, Chun-Wei David Chen, PhD4, Thomas Kindler, MD5*, Francis Burrows, PhD3*, Matthias Theobald, MD1*, Daniel Sasca, MD1*, Scott A. Armstrong, MD, PhD6,7 and Michael Kuehn, MD1*

1Department of Hematology, Medical Oncology, and Pulmonary Medicine, University Medical Center, Johannes Gutenberg-University, Mainz, Germany
2DFCI, Boston
3Kura Oncology, San Diego, CA
4Department of Systems Biology, Beckman Research Institute, Duarte, CA
5University Cancer Center of Mainz, Mainz, Germany
6Division of Hematology/Oncology, Boston Children's Hospital, Boston, MA
7Department of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, MA

MLL1-rearranged (MLL-r) leukemias are dependent on the interaction of the adaptor protein menin with the oncogenic MLL (KMT2A) fusion protein. We previously demonstrated that NPM1 mutant (NPM1mut) acute myeloid leukemias (AMLs) exhibit a similar dependency on the menin interaction with wildtype MLL, a histone 3 lysine 4 methyltransferase. Pharmacologic inhibition of the menin-MLL interaction reverses MEIS1/HOX-driven leukemic gene expression, induces differentiation, and has anti-leukemic activity in preclinical NPM1mut and MLL-r AML models. Ziftomenib is currently assessed in clinical phase I/II trials and explorative efficacy against relapsed / refractory AML has been demonstrated. Here, we characterized the effects of ziftomenib on MLL-r and NPM1mut AML in detail and systematically screened for synergistic drug combination partners.

First, we assessed the single-agent effects of ziftomenib on MLL-r (MOLM13, MV411, OCI-AML2) and NPM1mut (OCI-AML3) AML cell lines. Cellular assays demonstrated profound dose-dependent killing after seven days of treatment with IC50 values<25nM. HL60 and NB4 cells that are both wildtype for NPM1 and MLL served as negative controls and were not affected. Similar to previously characterized menin inhibitors (menin-i), ziftomenib uniformly induced transcriptional downregulation of MEIS1, PBX3, FLT3, and BCL2 in these leukemias, while genes upregulated upon ziftomenib were enriched for myelomonocytic differentiation processes. Ziftomenib-induced differentiation was also evidenced by cytology and upregulation of cell surface CD14 and CD11b.

To identify synergistic combinations, we designed a screen assessing single and combined effects of ziftomenib and 37 targeted compounds (selected by confirmed preclinical or clinical activity against AML), each at four different concentrations and in a constant 1:4 ratio. We observed significant synergistic effects (combination index<0.5) of ziftomenib in MLL-r and NPM1mut AML, particularly pronounced for agents targeting chromatin regulation & DNA damage (e.g., LSD1, PRMT5, and PARP) as well as apoptosis & cell cycle (e.g. BCL2, MCL1, and CDK4/6). As we have demonstrated with other menin-i, ziftomenib induced transcriptional downregulation of FLT3 in the FLT3-mutant AML setting and also exhibited synergistic activity with the FLT3 inhibitor gilteritinib. As ziftomenib also uniformly downregulated BCL2, we assessed its combination with venetoclax in more detailed response assays and found synergistic growth inhibition compared to single treatment or vehicle control in all tested MLL-r and NPM1mut AMLs. While OCI-AML3 cells are not sensitive to venetoclax, the combination with ziftomenib was synergistic when using prolonged combined agent exposure (5 days) but not with standard 24-48h venetoclax treatment. shRNA-based knockdown of MEN1 (menin) also sensitized MOLM13 to venetoclax. Next, we found by Annexin V staining that the combination enhanced apoptosis compared to single agent treatment. Also, BCL2 homology (BH) 3 profiling revealed that ziftomenib treated MLL-r and NPM1mut AML cells exhibited a significantly enhanced cytochrome c release in response to BIM, PUMA, BAD, and MS1 peptides. These findings suggest that ziftomenib-induced apoptotic priming may contribute to synergistic effects with venetoclax in these leukemias.

Next, we assessed the combination in a human MV411-derived leukemic xenograft model in vivo. We found significantly reduced leukemia burden defined by bone marrow engraftment after 14 days of treatment compared to the single agent or vehicle control animals. Single agent and combination assessment of four primary human NPM1mut AML patient samples in a human stroma cell co-culture model also showed the most pronounced anti-leukemic effects for the ziftomenib-venetoclax combination compared to all other controls.

In summary, we demonstrated that ziftomenib has significant activity against MLL-r and NPM1mut AML and synergizes with certain agents targeting chromatin regulation and apoptosis. The combination of ziftomenib with venetoclax is particularly synergistic and already available for clinical trial assessment.

Disclosures: Rausch: Abbvie: Other: Travel Support/expenses. Kessler: Kura Oncology: Current Employment, Current equity holder in publicly-traded company. Burrows: Kura Oncology: Current Employment, Current equity holder in publicly-traded company. Sasca: Abbvie: Honoraria; Astra-Zeneca: Honoraria; Bristol-Myers-Squibb: Honoraria; Biontech: Other: Current Employment (Spouse). Armstrong: Neomorph Inc: Consultancy; -: Patents & Royalties: MENIN inhibition WO/2017/132398A1; Syndax: Research Funding; Novartis: Research Funding; Janssen: Research Funding; Mana Therapeutics: Consultancy, Other: Shareholder; Twenty eight-seven Therapeutics: Consultancy, Other: Shareholder; Accent Therapeutics: Consultancy, Other: Shareholder; C4 Therapeutics: Consultancy, Other: SHareholder; Cyteir Therapeutics: Consultancy, Other: Shareholder; Imago Biosciences: Consultancy, Other: Shareholder. Kuehn: Abbvie: Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS / Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Daiichi Sankyo: Other: Travel Support; Gilead: Speakers Bureau; Jazz Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees; KURA-Oncology: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees.

*signifies non-member of ASH