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2778 Molecular and Clinical Features of FLT3 Juxtamembrane Domain Point Mutations (muts) in Acute Myeloid Leukemia (AML)

Program: Oral and Poster Abstracts
Session: 617. Acute Myeloid Leukemias: Biomarkers, Molecular Markers and Minimal Residual Disease in Diagnosis and Prognosis: Poster II
Hematology Disease Topics & Pathways:
Research, Acute Myeloid Malignancies, AML, Translational Research, Non-Biological therapies, genomics, Diseases, Therapies, Myeloid Malignancies, Biological Processes
Sunday, December 11, 2022, 6:00 PM-8:00 PM

Nadeen Anabtawi1*, Jessica Kohlschmidt, PhD2,3, Daelynn Buelow, PhD4*, Shelley Orwick, BS1*, Kennedy Coleman1*, Krzysztof Mrózek, MD, PhD5, Jonathan E. Kolitz, MD6,7, Bayard L. Powell, MD8, William G. Blum, MD9, Maria R. Baer, MD10, John C. Byrd, MD11, Wendy Stock, MD12, Geoffrey L. Uy, MD13, Ann-Kathrin Eisfeld, MD14,15, Sharyn Baker, PharmD PhD4 and James S. Blachly, MD16,17

1The Ohio State University Comprehensive Cancer Center, Columbus, OH
2Clara D. Bloomfield Center for Leukemia Outcomes Research, The Ohio State University Comprehensive Cancer Center, Columbus
3Alliance Statistics and Data Management Center, The Ohio State University Comprehensive Cancer Center, Columbus, OH
4Division of Pharmaceutics and Pharmacology, College of Pharmacy, Comprehensive Cancer Center, The Ohio State University, Columbus
5Clara D. Bloomfield Center for Leukemia Outcomes Research, The Ohio State University Comprehensive Cancer Center, Columbus, OH
6Monter Cancer Center, Zucker School of Medicine at Hofstra/Northwell, Lake Success, NY
7Monter Cancer Center, North Shore University Hospital and Long Island Jewish Medical Center, Northwell Health, New Hyde Park, NY
8Wake Forest Baptist Comprehensive Cancer Center, Winston Salem, NC
9Department of Hematology and Oncology, Winship Cancer Institute of Emory University, Atlanta, GA
10University of Maryland Greenebaum Comprehensive Cancer Center, Baltimore, MD
11University of Cincinnati College of Medicine, Cincinnati, OH
12University of Chicago, Chicago, IL
13Division of Oncology, Department of Medicine, Washington University School of Medicine, Saint Louis, MO
14Division of Hematology, Department of Internal Medicine, The Ohio State University Comprehensive Cancer Center, Columbus, OH
15Clara D. Bloomfield Center for Leukemia Outcomes Research, The Ohio State University, Columbus, OH
16James Comprehensive Cancer Center, The Ohio State University, Columbus, OH
17The Clara D. Bloomfield Center for Leukemia Outcomes Research, The Ohio State University Comprehensive Cancer Center, Columbus, OH

Background

The FLT3 gene encodes a transmembrane receptor normally expressed by hematopoietic stem or progenitor cells that when mutated plays a role in the development of AML. Mutations in FLT3 are found in ~ 30% of newly diagnosed AML cases and primarily occur as internal tandem duplication (ITD) within the juxtamembrane domain (JMD) of the receptor (~ 25%) or point muts in the tyrosine kinase domains (TKD; 7–10%). Recently, point muts in the JMD have been identified as additional recurrent events. The JMD has an auto-inhibitory role preventing dimerization of FLT3 receptor, and disruption of this key region, whether by ITD or point mutation, is expected to result in constitutive activation of FLT3 receptor. However, thus far little is known about the biologic and clinical significance of FLT3-JMD.

Methods

To evaluate biologic and prognostic impacts of FLT3-JMD muts, we analyzed 1658 patients (pts) with newly diagnosed de novo AML treated similarly on frontline CALGB/Alliance protocols. All pts underwent high-throughput sequencing analysis of 80 AML-associated genes including FLT3 JMD and TKD regions, enabling FLT3-JMD variant curation. Survival analyses were conducted for pts with JMD present at ≥5% variant allele frequency (VAF). We tabulated distinct JMD muts and produced expression constructs for a subset of these. FLT3-JMD constructs were lentivirally transduced into Ba/F3 cells and cell growth was assessed over 7 days in the absence of interleukin 3 (IL-3). The analyses of the effect of FLT3 inhibitors on signaling and cell viability were done using MTT assays to evaluate the sensitivity of FLT3-JMD muts to available treatment options.

Results

We identified 44 point muts and deletions within FLT3-JMD in 39 pts, a prevalence of 2.4%. Most common were amino acid exchanges at residue V592 (D/A/I; n=10), followed by Y597C/*/del (n=4). JMD alterations frequently co-occurred with FLT3-ITD and TKD muts: of 39 pts, 26 (67%) also had ITD and 11 (28%) also had TKD. NPM1, RUNX1 and ASLX1 were the most frequently co-mutated genes.

Considering pts with VAF ≥5% (n=29), relapse rate (RR) was similar (62 vs 48%, p=0.55) but disease-free survival (DFS) trended shorter (median, 1.8 vs 2.4 y, p=0.07) in pts having FLT3-JMD with or without TKD (n=16) vs pts with TKD alone (n=124; p<.001), while both did significantly better than pts bearing ITD (RR 73%, DFS, median, 0.6 y). Despite this, median overall survival of pts with FLT3-JMD±TKD was 3.4 y compared to 1.7 y in FLT3-TKD only, although no significant difference existed at 5 y (44 vs 37%, p=0.19).

To complement these observations and explore biologic consequences of FLT3-JMD muts, we evaluated the transforming potential of the most frequent JMD muts in Ba/F3 cells, and their sensitivity to a panel of approved and clinical candidate FLT3 inhibitors in cell survival and signaling assays. Point muts in the FLT3-JMD were found to have transforming potential, because Ba/F3 cells expressing Y572C, V579A and V592A in the FLT3-JMD were able to grow in the absence of IL-3. Cell survival assays revealed that FLT3-JMD point muts can be targeted by available FLT3 inhibitors. Compared with FLT3-ITD, Y572C, V592A and V592A conferred high sensitivity to gilteritinib (Fig. 1A), in addition to a panel of available FLT3 inhibitors (Fig. 1B). Notably, JMD muts were also sensitive to quizartinib and sorafenib, whereas TKD muts are not.

Discussion

We analyzed a new and relatively understudied class of activating FLT3 muts clustering in the JMD and having outcome and treatment implications. Occurrence of JMD overall is rare, and co-occurrence with other FLT3 alterations is common, complicating dissection of outcomes. Patient data in a large historical dataset suggest that outcomes of pts with FLT3-JMD are similar to those with TKD, although with the possibility of higher RR and shorter DFS. Their median OS appeared longer than that of pts with TKD alone, but with no OS difference at later timepoints. These findings, if corroborated by larger studies, suggest distinct relapse dynamics. In vitro, the most common FLT3-JMD point muts confer a transforming potential to hematopoietic cells as predicted, and our data suggest that pts carrying FLT3-JMD point muts could respond to treatment with available FLT3 inhibitors. Finally, in contrast to TKD, JMD mutant cells are sensitive to type 2 FLT3 inhibitors, expanding the scope of treatment options for these pts.

Disclosures: Powell: Ambit Biosciences: Research Funding; Genentech: Research Funding; Hoffman LaRoche: Research Funding; Jazz Pharmaceuticals: Research Funding; Novartis: Research Funding; Pfizer: Research Funding; Rafael Pharmaceuticals: Consultancy, Research Funding. Baer: Forma: Research Funding; AbbVie: Research Funding; Kura Oncology: Research Funding; Ascentage: Research Funding; Takeda: Research Funding; Kite: Research Funding. Byrd: Janssen: Consultancy; Kura: Consultancy; Newave: Consultancy; Syndax: Consultancy; AstraZeneca: Consultancy; Novartis: Consultancy; Vincerx: Consultancy, Current equity holder in private company, Current equity holder in publicly-traded company, Current holder of stock options in a privately-held company; Zencor: Research Funding; Pharmacyclics: Research Funding; Ohio State University: Patents & Royalties; AbbVie: Consultancy; Trillium: Consultancy. Stock: Newave Pharmaceuticals: Consultancy; Syndax: Consultancy, Honoraria; Servier: Honoraria; Pluristem: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria, Research Funding; Kura Oncology: Honoraria; Kite: Honoraria; Jazz Pharmaceuticals: Honoraria; Amgen: Honoraria. Eisfeld: Karyopharm Therapeutics: Other: Spouse is current company employee. Blachly: KITE Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees; INNATE Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Consultancy, Membership on an entity's Board of Directors or advisory committees; AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Astellas: Consultancy, Membership on an entity's Board of Directors or advisory committees; MingSight Pharmaceuticals: Research Funding.

*signifies non-member of ASH