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2785 BCL2A1: A Novel Target in Refractory Acute Myeloid Leukemia with FLT3-ITD/D835 Dual Mutations

Program: Oral and Poster Abstracts
Session: 604. Molecular Pharmacology and Drug Resistance in Myeloid Diseases: Poster III
Hematology Disease Topics & Pathways:
Biological, AML, Diseases, Therapies, Myeloid Malignancies, TKI
Monday, December 7, 2020, 7:00 AM-3:30 PM

Kotoko Yamatani, MD1,2*, Tomohiko Ai, MD, PhD2*, Kaori Saito2*, Haeun Yang2*, Vivian Ruvolo, MS3*, Po Yee Mak3*, Sonoko Kinjo, PhD4*, Kazuho Ikeo, PhD4*, Hironori Harada, MD, PhD5, Kazuhiro Katayama, PhD6*, Takashi Miida, MD, PhD2*, Marina Konopleva, MD, PhD7, Weiguo Zhang, MD, PhD8, Bing Z Carter, PhD9, Yoshihide Hayashizaki, MD, PhD10*, Michael Andreeff, MD, PhD3 and Yoko Tabe, MD, PhD2,11

1Research Fellow of the Japan Society for the Promotion of Science, Tokyo, Japan
2Department of Clinical Laboratory Medicine, Graduate School of Medicine, Juntendo University, Tokyo, Japan
3Section of Molecular Hematology and Therapy, Department of Leukemia, The University of Texas MD Anderson Cancer Center, Houston, TX
4Centers for Information Biology, National Institute of Genetics, Shizuoka, Japan
5Department of Hematology, Graduate School of Medicine, Juntendo University, Tokyo, Japan
6Laboratory of Molecular Targeted Therapeutics, School of Pharmacy, Nihon University, Chiba, Japan
7Department of Leukemia, University of Texas, MD Anderson Cancer Center, Houston, TX
8Section of Molecular Hematology and Therapy, Department of Leukemia, U.T.M.D. Anderson Cancer Center, Houston, TX
9Section of Molecular Hematology and Therapy, Department of Leukemia, The University of Texas M.D. Anderson Cancer Center, Houston, TX
10Preventive Medicine & Diagnosis Innovation Program, RIKEN Center for Life Science Technoligies, Kanagawa, Japan
11Section of Molecular Hematology and Therapy, Department of Leukemia, MD Anderson Cancer Center, Houston, TX

Internal tandem duplications in the juxtamembrane domain of FMS-like tyrosine kinase 3 gene (FLT3-ITD) and missense mutations in the gene’s tyrosine-kinase domain (FLT3-TKD) play critical roles in the pathophysiology of acute myeloid leukemia (AML). Recent study revealed that AML cells resistant to quizartinib, a type II TKI, consist of heterogeneous clonal populations harboring wild-type FLT3 as well as FLT3-ITD, FLT3-TKD and FLT3-ITD/TKD mutations, and on- and off-target mechanisms may contribute to the resistance to TKIs. To overcome the heterogeneous resistance mechanisms of FLT3-ITD and TKD mutations, various combinatorial therapies have been investigated. For example, BCL-2 inhibition in the presence of TKIs increased survival a murine FLT3-ITD AML model, and a phase Ib/II clinical trial of a combination of quizartinib with venetoclax, a BCL2 inhibitor is ongoing in relapsed/refractory FLT3-mutant AML patients (NCT03735875).

To dissect underlying mechanisms of drug-resistance and exploring new targets in refractory AML with FLT3-ITD and TKD mutations, we investigated alterations of transcriptome signatures by analyzing AML samples with FLT3-ITD/D835 dual mutations. Previously, we reported BCL2A1 transcriptomes were upregulated in primary AML cells with FLT3-ITD/D835 dual mutations compared to cells with FLT3-ITD mutations only. This was recapitulated in the MV4-11 cells harboring FLT3-ITD/D835 dual mutations after 6 month-exposure to quizartinib. The MV4-11 cells with the FLT3-ITD/D835 dual mutations became resistant to quizartinib, and the cells also became resistant to venetoclax, a BCL2 inhibitor (Yamatani et al. ASH 2019).

In this study, we further investigated BCL2A1 as new target in refractory AML with FLT3-ITD/D835 dual mutations. First, we examined whether overexpression of BCL2A1 induces drug-resistance in MV4-11 and Molm13 cell lines with FLT3-ITD. While parental MV4-11 and Molm13 cells are sensitive to venetoclax and quizartinib, MV4-11 and Molm13 cells transfected with lentivirus carrying BCL2A1 became resistant to venetoclax (IC50: MV4-11 with BCL2A1 over-expression >1000 nM vs. mock vector 0.71 nM; Molm13 with over-expression >1000 nM vs. mock vector 0.38 nM, 72h). In contrast, the sensitivity to quizartinib was retained in the BCL2A1 overexpressing MV4-11 and Molm13 cells. These findings indicate that the overexpression of BCL2A1 could play a role in the acquired resistance to venetoclax, but not to quizartinib.

Bromodomain-containing protein 4 (BRD4), a family member of bromodomain and extra-terminal motif (BET) is known to transcriptionally modulate BCL2A1 gene expression. Next, we examined effects of CPI-0610, a BET inhibitor, on MV4-11 cells with FLT3-ITD or the FLT3-ITD/D835 dual mutation. CPI-0610 inhibited cell growth of MV4-11 cells by inducing apoptosis irrespective of co-existing FLT3 mutations (IC50: FLT3-ITD/D835, 255 nM vs. FLT3-ITD, 191 nM, 72h). Immunoblotting analyses confirmed that BET inhibition by CPT-0610 decreased the expression of BCL2A1 in MV4-11 cells FLT3-ITD/D835.

In conclusion, transcriptome analysis and molecular pharmacological approaches identified alterations in the anti-apoptotic BCL2 family proteins in double-mutant FLT3 leukemias. BCL2A1 upregulation might be involved in drug resistance of FLT3-ITD/D835 dual mutant AML cells, and could be a promising new target in refractory AML with FLT3-ITD/D835 dual mutations.

Disclosures: Konopleva: Cellectis: Research Funding; Eli Lilly: Research Funding; Calithera: Research Funding; Sanofi: Research Funding; Ascentage: Research Funding; AstraZeneca: Research Funding; Agios: Research Funding; Stemline Therapeutics: Consultancy, Research Funding; Kisoji: Consultancy; Genentech: Consultancy, Research Funding; F. Hoffmann La-Roche: Consultancy, Research Funding; Forty-Seven: Consultancy, Research Funding; AbbVie: Consultancy, Research Funding; Reata Pharmaceutical Inc.;: Patents & Royalties: patents and royalties with patent US 7,795,305 B2 on CDDO-compounds and combination therapies, licensed to Reata Pharmaceutical; Rafael Pharmaceutical: Research Funding; Ablynx: Research Funding; Amgen: Consultancy. Carter: Syndax: Research Funding; AstraZeneca: Research Funding; Amgen: Research Funding; Ascentage: Research Funding. Andreeff: Daiichi-Sankyo; Breast Cancer Research Foundation; CPRIT; NIH/NCI; Amgen; AstraZeneca: Research Funding; Centre for Drug Research & Development; Cancer UK; NCI-CTEP; German Research Council; Leukemia Lymphoma Foundation (LLS); NCI-RDCRN (Rare Disease Clin Network); CLL Founcdation; BioLineRx; SentiBio; Aptose Biosciences, Inc: Membership on an entity's Board of Directors or advisory committees; Amgen: Research Funding; Daiichi-Sankyo; Jazz Pharmaceuticals; Celgene; Amgen; AstraZeneca; 6 Dimensions Capital: Consultancy.

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