-Author name in bold denotes the presenting author
-Asterisk * with author name denotes a Non-ASH member
Clinically Relevant Abstract denotes an abstract that is clinically relevant.

PhD Trainee denotes that this is a recommended PHD Trainee Session.

Ticketed Session denotes that this is a ticketed session.

933 Multi-Omic Single-Cell Sequencing Reveals Genetic and Immunophenotypic Clonal Selection in Patients With FLT3-Mutated AML Treated With Gilteritinib/Venetoclax

Program: Oral and Poster Abstracts
Type: Oral
Session: 604. Molecular Pharmacology and Drug Resistance: Myeloid Neoplasms: Immune Signaling and Antibody-therapeutic Targeting in Myeloid Neoplasms
Hematology Disease Topics & Pathways:
Combination therapy, genomics, Therapies, Biological Processes, Technology and Procedures, molecular testing, omics technologies
Monday, December 12, 2022: 5:00 PM

Vanessa E. Kennedy, MD1, Cheryl Peretz, MD2, Paul Lee, MD, PhD3*, Brenda Chyla, PhD4, Yan Sun, PhD4*, Monique Dail, PhD5*, Jason Hill, PhD6*, Elaine Tran1*, Andrew Koh1*, Jessica K. Altman, MD7, Naval Daver, MD8, Mark J. Levis, MD9, James McCloskey, MD10*, Alexander Perl, MD11 and Catherine Smith, MD1

1University of California San Francisco, San Francisco, CA
2Department of Pediatrics and Helen Diller Family Comprehensive Cancer Center, University of California San Francisco, San Francisco, CA
3AbbVie, Inc., North Chicago, IL
4AbbVie Inc., North Chicago, IL
5Genentech Inc., South San Francisco, CA
6Astellas Pharma Global Development, Northbrook, IL
7Department of Medicine, Northwestern University Feinberg School of Medicine, Chicago, IL
8Department of Leukemia, The University of Texas MD Anderson Cancer Center, Houston, TX
9Division of Hematologic Malignancies, Johns Hopkins University, Baltimore, MD
10John Theurer Cancer Center, Hackensack University Medical Center, Hackensack, NJ
11Division of Hematology-Oncology, University of Pennsylvania School of Medicine, Philadelphia, PA

Background: The combination of the FLT3 inhibitor gilteritinib with the BCL-2 inhibitor venetoclax (VenGilt) is highly efficacious in relapsed/refractory (R/R) FLT3-mutated acute myeloid leukemia (AML). In a recent phase 1b trial, VenGilt demonstrated a modified complete response rate of 75% (Daver et al, J Clin Oncol. 2022; published online). However, many patients (pts) relapsed after VenGilt. Proposed resistance mechanisms include RAS pathway mutations (Ven and Gilt) and monocytic immunophenotype (Ven). Using single cell (SC) multi-omic profiling, we sought to identify drivers of resistance to this combination.

Methods: We analyzed 35 serial samples from 12 pts with R/R AML treated with VenGilt on the phase 1b trial (NCT03625505). We performed high-throughput SC DNA sequencing (Mission Bio Tapestri) on all samples and concurrent multi-omic SC cell-surface immunophenotyping on 18 samples.

Results: We analyzed 107,528 cells (median 8960 cells/pt) from 12 pts with 2–5 serial timepoints. Eight pts had previously received tyrosine kinase inhibitors (TKI). Nine pts had clinical responses to VenGilt with a median relapse-free time of 8.3 mo. Of these 9 pts, 3 proceeded to transplant.

Each pt had ≥1 genetically distinct clone identified (median 4.5, range 2–6). Fourteen FLT3mut clones were detected, with 2 pts having 2 separate FLT3mut clones. In all 12 pts, the FLT3mut clones were reduced with VenGilt. In 7 pts with a total of 9 clones, the FLT3mut population decreased to an undetectable level. Three pts had FLT3-IDH1/2 co-mutated clones, and the double mutant clones were reduced to undetectable levels in all 3. One pt developed a dominant IDH2mut clone at relapse without a FLT3 co-mutation. Nine pts demonstrated an initial decrease in non-FLT3mut clones, including clones with DNMT3A, NPM1, IDH1/2, TET2, EZH2, WT1, and NRAS mutations, suggesting some activity of VenGilt against non-FLT3mut populations. In 3 pts, all clones aside from those associated with clonal hematopoiesis of indeterminant potential (CHIP) were undetectable at follow-up.

Five pts had ≥1 pre-existing NRAS- or KRAS-mutated clone. In 4/5 pts, the RASmut clone expanded on therapy and was dominant at relapse in 3/4 pts, with the fourth pt having a dominant IDH2mut clone. All 4 pts with expanding RASmut clones had prior TKI, while the pt whose RASmut clone decreased did not. Pre-existing RAS populations were small (0.001–18.1% of total cells) and were undetectable by bulk sequencing in 2/4 cases. RAS was not co-mutated with FLT3 in the same single cell in any cases. In 2 pts, FLT3mut clones present before VenGilt initially decreased but subsequently expanded on therapy. No de novo mutations within resistant/expanding FLT3mut clones were detected.

We demonstrated immunophenotypic evolution for individual genotypes, including both FLT3mut and FLT3WT clones. For 5 pts with serial multi-omic SC samples, increased CD11b and CD13 expression was observed over time on a single cell basis in ≥1 genotype within individuals, suggesting increased monocytic-like features. These features were observed among 4 RASmut, 2 FLT3mut, and 1 TET2mut clones. At baseline, these 5 pts had heterogenous immunophenotypic profiles, with genetic clones occupying monocytic, immature, and mixed phenotypes; however, all 5 pts demonstrated ≥1 clone with increased monocytic immunophenotypic features after VenGilt treatment.

Conclusion: Multi-omic SC sequencing enabled direct measurement of genetic and immunophenotypic clonal architecture and evolution with VenGilt treatment. VenGilt was highly effective against FLT3-mutated clones, even in the presence of co-mutations. Genetic resistance was associated with outgrowth of pre-existing subclones, primarily RAS mutations, rather than acquistion of de novo mutations. This differs from prior studies of Gilt monotherapy, in which both outgrowth of parallel clones and secondary mutations within the FLT3-mutated clone were associated with resistance. These data suggest the addition of Ven may suppress secondary resistance mutations, possibly due to enhanced apoptosis and cytotoxicity against FLT3-mutated cells. Multiple clones, predominatly those with a RAS mutation, developed monocytic features under VenGilt pressure. It is unknown whether monocytic immunophenotype is an independent resistance mechanism or a bystander to other mechanisms, such as increased MCL1 or RAS pathway gene expression.

Disclosures: Lee: Abbvie: Current Employment, Current equity holder in publicly-traded company. Chyla: AbbVie: Current Employment, Current holder of stock options in a privately-held company. Sun: AbbVie: Current Employment, Current equity holder in publicly-traded company. Dail: Roche: Current equity holder in publicly-traded company; Genentech: Current Employment. Hill: Astellas Pharma Inc.: Current Employment; Ligacept, LLC: Current equity holder in private company; Targeted Molecular Diagnostics, Ligacept: Patents & Royalties: Patents: US 7862995, US 9051388, US 9683222. Altman: AbbVie, Astellas: Consultancy; GlycoMimetics: Other: Data monitoring committee member; AbbVie: Consultancy, Honoraria, Research Funding; Astellas: Consultancy, Honoraria, Research Funding; ALX Oncology, Amgen, Aprea, Aptos, Bristol Myers Squibb, Boehringer Ingelheim, Celgene, Fujifilm, Immunogen, Kartos, Loxo: Research Funding; BioSight: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: reimbursement for travel, Research Funding; Kura Oncology: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Syros Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees. Daver: Kartos and Jazz Pharmaceuticals: Other: Data monitoring committee member; Agios, Celgene, SOBI and STAR Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Karyopham Therapeutics and Newave Pharmaceutical: Research Funding; Astellas, AbbVie, Genentech, Daiichi-Sankyo, Novartis, Jazz, Amgen, Servier, Karyopharm, Trovagene, Trillium, Syndax, Gilead, Pfizer, Bristol Myers Squibb, Kite, Actinium, Arog, Immunogen, Arcellx, and Shattuck: Consultancy, Other: Advisory Role; Astellas, AbbVie, Genentech, Daiichi-Sankyo, Gilead, Immunogen, Pfizer, Bristol Myers Squibb, Trovagene, Servier, Novimmune, Incyte, Hanmi, Fate, Amgen, Kite, Novartis, Astex, KAHR, Shattuck, Sobi, Glycomimetics, Trillium: Research Funding. Levis: AbbVie, Amgen, Astellas, Bristol Myers Squibb, Daiichi-Sankyo, FujiFilm, Jazz Pharmaceuticals, and Menarini: Consultancy; Astellas, and FujiFilm: Research Funding. McCloskey: AbbVie, CTI BioPharma, and Novartis: Consultancy; AbbVie, Amgen, Bristol Myers Squibb, Incyte, Jazz Pharmaceuticals, Stemline, and Takeda: Speakers Bureau. Perl: Astellas, Abbvie, Daiichi Sankyo, FujiFilm, Syndax: Research Funding; Astellas, Daiichi Sankyo, Abbvie, Genentech, BerGenBio, Immunogen, BMS/Celgene, Actinium: Membership on an entity's Board of Directors or advisory committees; Astellas, Daiichi Sankyo, AbbVie, Forma, Sumitomo Dainippon, BeatAML LLC: Consultancy. Smith: Astellas: Membership on an entity's Board of Directors or advisory committees; Genentech: Membership on an entity's Board of Directors or advisory committees; Revolution Medicines: Research Funding; Abbvie: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Research Funding; Erasca: Research Funding.

*signifies non-member of ASH