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3854 Unraveling at Diagnosis Chemoresistant Relapse-Initiating Clones in Acute Myeloid Leukemia: Combined Functional and Genomic Analyses Using Single-Cell-Derived Hematopoietic Stem and Progenitor Cell Subpopulations

Program: Oral and Poster Abstracts
Session: 501. Hematopoietic Stem and Progenitor Cells and Hematopoiesis: Basic and Translational: Poster III
Hematology Disease Topics & Pathways:
Research, Translational Research
Monday, December 12, 2022, 6:00 PM-8:00 PM

Tsila Zuckerman, MD1,2, Tal Gabay, PhD1*, Nofar Stolero1*, Niv Rabhun1*, Rawan Sabah, PhD1*, Ofir Raz3*, Yaara Neumeier3*, Zipora Marx3*, Liming Tao, PhD3*, Tamir Biezuner, PhD4*, Shiran Amir3*, Rivka Adar3*, Ron Levy3*, Noa Chapal Ilani, PhD4*, Liran I. Shlush, MD, PhD4, Ehud Shapiro, PhD3* and Shlomit Yehudai-Reshef, PhD1,2*

1Department of Hematology and Bone Marrow Transplantation, Rambam Health Care Campus, Haifa, Israel
2The Ruth and Bruce Rappaport Faculty of Medicine, Technion, Haifa, Israel
3Department of Computer Science and Applied Mathematics, Weizmann Institute of Science, Rehovot, Israel
4Department of Immunology, Weizmann Institute of Science, Rehovot, Israel

Acute myeloid leukemia (AML) emerges from abnormal hematopoietic stem/progenitor cells (HSPCs). While most patients achieve complete remission, about 60% of them experience disease relapse. AML is characterized by genetically heterogeneous cell subpopulations with different properties, allowing certain leukemic HSPC subsets to evade treatment and/or acquire new mutations promoting their survival. Both these mechanisms could be involved in disease relapse.

The rationale of the current research was to identify at diagnosis the leukemic HSPC clones that could promote AML progression, aiming to therapeutically target them at an early disease stage, thus preventing relapse. To that end, this study explored the genetic composition, chemoresistance propensity and leukemogenic potential of single HSPCs derived from AML patients.

Single leukemic cells (SLCs) obtained from AML patients at diagnosis and relapse were sorted into 4 HSPC subpopulations (HSC-MPP, MLP, CMP-MEP and GMP). Additionally, single HSPC-derived subclones were generated. Both SLCs and the subclones were analyzed for their chemoresistance, leukemia-associated mutations and cell lineage, using next generation sequencing, immunophenotyping, and lineage analyses with phylogenetic tree reconstruction. An NSG-mouse model was used in chemoresistance screening and functional assays to assess HSPC leukemogenic potential. HSPCs obtained from healthy donors (HD) were employed as control.

The most prevalent components of the AML-HSPC subpopulations identified at diagnosis were MLP33-/+-like cells of the lymphoid lineage and GMP33+-like cells of the myeloid linage, while HSC-MPP33-like cells appeared to be the major component of HD-HSPCs. Notably, either GMP-like (granulocyte/monocyte progenitor) or MLP-like (multipotent lymphoid progenitor) HSPC subpopulation was also shown to be most frequent in all the samples obtained at relapse, suggesting significant involvement of these HSPC subsets in AML progression.

Ex-vivo exposure of HSPC subpopulations to various concentrations of chemotherapy agents, cytarabine (Ara-C) or daunorubicin, displayed diverse response. The viability of the GMP33+-like fraction obtained at diagnosis was unaffected by these agents, irrespective of their concentration. Conversely, the diagnosis-derived MLP33+-like fraction was found to be chemo-sensitive. Moreover, the sensitivity of the latter cell fraction to these agents was different, with the cells derived from 2/10 AML patients being sensitive to Ara-C and not to daunorubicin, while cells obtained from another 2/10 patients demonstrated reverse results. In samples derived from additional 3/10 patients, GMP-like and MLP-like fractions appeared to be sensitive to both agents.

Phylogenetic tree reconstruction combined with single cell/subclone genetic analyses demonstrated 2 distinct clusters present at diagnosis and relapse in all the evaluated AML patients. In 2 patients, the same GMP-like and MLP-like subpopulations were detected both at diagnosis and relapse, suggesting their contribution to disease progression, while in an additional patient the relapse clearly originated from the MLP-like subpopulation only. The leukemogenic potential of chemoresistant GMP-like and MLP-like subpopulations was further confirmed in an NSG mouse model.

The comprehensive strategy, combining single-cell analysis and phylogenetic assessment of AML-HSPC subpopulations, employed in this study, enabled advanced functional and genomic profiling of leukemic HSPCs, that unraveled their inter- and intra-patient heterogeneity. The presence of GMP-like and/or MLP-like subpopulations at AML diagnosis, their identified chemoresistance propensity and occurrence at relapse, strongly point to the involvement of these HSPC fractions in relapse initiation. Overall, these findings highlight the need for personalized therapy targeting specific HSPC subsets as a therapeutic approach to prevent AML recurrence.

Disclosures: Zuckerman: Orgenesis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; BioSight Ltd: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Cellect Biotechnology: Honoraria, Speakers Bureau; Janssen: Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau.

*signifies non-member of ASH