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210 Disrupting Mitochondrial Dynamics and Metabolism in Leukemic Stem Cells through Mitochondrial PCNA Inhibition: The Role of AOH1996

Program: Oral and Poster Abstracts
Type: Oral
Session: 604. Molecular Pharmacology and Drug Resistance: Myeloid Neoplasms: Novel Approaches to Targeting Epigenetics, Apoptosis and Immune Effectors in Myeloid Malignancies
Hematology Disease Topics & Pathways:
Research, Acute Myeloid Malignancies, AML, Combination therapy, Translational Research, Drug development, Diseases, Treatment Considerations, Metabolism, Myeloid Malignancies, Biological Processes
Saturday, December 7, 2024: 3:15 PM

Hyunjun Kang, PhD1*, Melissa Valerio2*, Jia Feng3*, Long Gu, PhD4*, Dinh Hoa Hoang2*, Shawn Sharkas5*, Khyatiben V. Pathak6*, Jennifer Jossart, PhD4*, Yuriy Shostak, PhD7*, Zhuo Li, PhD8*, Bin Zhang5, Patrick Pirrotte6*, Jefferson Perry, PhD4*, Robert Hickey, PhD9*, Linda H Malkas, PhD4*, Guido Marcucci10 and Le Xuan Truong Nguyen6,11*

1Department of Hematologic Malignancies Translational Science, City of Hope National Medical Center, Duarte, CA
2Department of Hematologic Malignancies Translational Science, Gehr Family Center for Leukemia Research, City of Hope National Medical Center and Beckman Research Institute, Duarte, CA
3Department of Hematologic Malignancies Translational Science, Beckman Research Institute and City of Hope National Medical Center, Duarte, California, USA, Duarte
4Department of Molecular Diagnostics & Experimental Therapeutics, Beckman Research Institute of City of Hope, Duarte, CA, USA, Duarte
5Department of Hematological Malignancies Translational Science, Gehr Family Center for Leukemia Research, City of Hope Medical Center and Beckman Research Institute, Duarte, CA
6Cancer & Cell Biology Division, Translational Genomics Research Institute, Phoenix, AZ
7City of Hope Medical Center, Monrovia, CA
8Beckman Research Institute, City of Hope National Medical Center, Duarte, California, USA, Duarte
9Department of Cancer Biology & Molecular Medicine, Beckman Research Institute of City of Hope, Duarte, CA, USA, Duarte
10Department of Hematologic Malignancies Translational Science, Duarte, CA
11Department of Hematologic Malignancies Translational Science, Gehr Family Center for Leukemia Research, City of Hope National Medical Center and Beckman Research Institute, South El Monte, CA

Proliferating cell nuclear antigen (PCNA) plays a crucial role in DNA synthesis and repair and is a marker of tumor progression. Different from its nuclear counterpart, cytoplasmic PCNA plays a role in mitochondrial DNA maintenance, dynamics regulation, and cellular stress responses. Cytoplasmic PCNA is highly expressed in acute myeloid leukemia (AML) cells, especially in leukemia stem cells (LSCs), supporting their oxidative metabolism and growth. LSCs depend on mitochondrial fusion (mitofusion), fatty acid oxidation (FAO), and oxidative phosphorylation (OXPHOS) for survival. Thus, given the PCNA role in regulation of mitochondrial dynamic and metabolism, targeting cytoplasmic PCNA may severely affect LSCs’ homeostasis, resulting in elimination of these cells and possibly disease eradication.

Building on the previous PCNA inhibitor version AOH1160 (PMID: 29967249), we designed AOH1996 (AOH), an improved analog that is orally administrable and metabolically stable. AOH effectively inhibits tumor growth and induces DNA damage in multiple cancer cells, with minimal toxicity in animal studies (PMID: 37531956). In this study, we showed that AOH significantly inhibited cell growth and induced apoptosis in AML cell lines and primary CD34+CD38- blasts (enriched for LSCs), while sparing normal CD34+CD38- cells (enriched for hematopoietic stem cells). AOH treatment in primary CD34+CD38- AML blasts significantly suppress FAO/OXPHOS levels and reduces mitochondria size (DMSO control: 1.1 μm, AOH: 0.7 μm), suggesting AOH inhibits mitofusion, leading to metabolic suppression. Mechanistically, we demonstrated that mitochondrial PCNA binds to the mitofusion-regulated OPA1 protein through the AIMP motif in LSCs. Adding AOH disrupts this interaction, leading to increased OPA1 binding with the E3 ligase MARCH5, which results in OPA1 ubiquitination and degradation in these cells. The inhibition of PCNA binding and the subsequent loss of OPA1 stability caused by AOH lead to decreased mitofusion and reduced FAO/OXPHOS levels, resulting in the inhibition of LSC growth. In vivo, AOH (100 mg/kg, PO, BID, 3 weeks) significantly reduced peripheral blood (PB) leukemic (hCD45+) engraftment rate (0.894%) in FLT3-WT transplanted Es1(ko) SCID PDX mice compared to vehicle (VEH; 3.812%; p<0.001) and prolonged survival [median survival (MS): 50 vs 35 days, p<0.0001]. Furthermore, in secondary transplant experiments recipients of bone marrow (BM) from AOH-treated donors survived longer that BM recipients from VEH-treated donors (MS: 42 vs 30 days, p<0.0001), indicating that AOH treatment reduced LSC burden.

The BCL-2 inhibitor venetoclax (VEN) has been shown to impact on the oxidative metabolism that supports LSC homeostasis. Given the AOH inhibition of mitochondrial PCNA-regulated mitofusion and FAO/OXPHOS in LSCs, we hypothesized a synergism between AOH and VEN. We showed that the AOH and VEN in combination synergistically decreased FAO/OXPHOS and mitofusion, and increased apoptosis of LSCs. For in vivo experiments, we transplanted the AML MllPTD/WT/Flt3ITD/ITD BM cells into Ces1c(ko) B6 AML mice and treated each group (n=10) with VEH (100 mg/kg, BID, PO), AOH (100 mg/kg, BID, PO), VEN (100 mg/kg, daily, PO), or VEN/AOH (same dosages as single agents) for 3 weeks. Compared with VEH or single agents, VEN/AOH significantly reduced PB leukemic engraftment and prolonged survival of primary transplanted mice (MS: VEN/AOH 54 vs VEN 34, p<0.0001; vs AOH 43.5, p=0.0006; vs VEH 34 days, p<0.0001) and secondary transplanted mice [MS: VEN/AOH 49.5 vs VEN 32.5, p<0.0001; vs AOH 41, p=0.006; vs VEH 28 days, p<0.0001]. Similar results were also observed in FLT3-WT transplanted Es1(ko) SCID PDX mice, with significantly prolonged survival in primary transplanted mice treated with VEN/AOH compared to single agents or VEH (MS: VEN/AOH 75.5 vs VEN 48, p<0.0001; vs AOH 55, p=0.0006; vs VEH 41 days, p<0.0001); and in secondary transplanted mice (MS: VEN/AOH 76 vs VEN 51, p<0.0001; vs AOH 60, p=0.0024; vs VEH 40 days, p<0.0001).

In summary, AOH1996 has a potent antileukemic activity in AML models through inhibition of mitochondrial PCNA-regulated mitochondria dynamics and metabolism and decrease of LSC burden. We demonstrated a better activity when AOH1996 is combined with VEN. While VEN is FDA-approved for AML, AOH1996 is in clinical trials for solid tumors.

Disclosures: Perry: RLL LLC: Other: stakeholder; Neumora Therapeutics Inc: Other: stakeholder; WinSanTor Inc: Other: stakeholder.

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