Characterization of Dysregulated Transcription in TP53-Mutated AML Subpopulations

Introduction: TP53 is a DNA binding protein that regulates transcription and exerts many functions critical to homeostasis. AML with mutated TP53 (TP53mt) is associated with dismal outcomes and resistance to all currently available therapies. Therefore, delineation of transcriptional dysregulation in TP53mt AML is critical for identification of vulnerabilities in this leukemia subtype. Leukemia cells are functionally heterogeneous, with many cells at diagnosis susceptible to chemotherapy, and a minority of resistant cells that persist despite treatment. Deeper understanding of all leukemia sub-populations is necessary for elucidating mechanisms of resistance. We hypothesized that sub-populations such as leukemia-stem cells (LSCs), and therapy-residual cells possess identifiable, targetable characteristics that drive resistance. We performed RNA-sequencing and compared differences in gene expression between these sub-populations.

Methods: We collected 113 bone marrow samples from 39 patients (pts) with adverse risk AML, including 16 pts with TP53mt (41%), 14 pts with FLT3 mutations (FLT3mt), and 10 pts (26%) with other adverse risk. We performed RNA-sequencing on paired pre- and post-treatment, bulk and FACS-sorted samples. Mononuclear cells were flow-sorted for bulk (CD45dim) and LSCs (Lin-CD34+CD38-CD123+) from diagnostic samples. Post-treatment samples were sorted for bulk mononuclear cells and therapy-residual cells, determined based on patient-specific aberrant phenotype using multi-color flow cytometry analysis (Xu J et al., Clinics in laboratory medicine 2017). Samples with low-expression of housekeeping genes were excluded from the analysis. Differential expression was analyzed using DESeq2 and Gene Set Enrichment Analysis (GSEA) was performed using the HALLMARK gene set and considered statistically significant if q<0.1.

Results: Regardless of genetic context, we found that LSCs were characterized by down regulation of immune-related pathways such (interferon-g response, allograft rejection, inflammatory response), and upregulation of oxidative phosphorylation and DNA repair among top dysregulated pathways when compared with bulk cells. Unlike FLT3mt, TP53mt LSCs had upregulation of MYC targets when compared with bulk cells. LSCs with TP53mt had a characteristic gene expression signature with a consistent upregulation of 11 genes (Figure 1). Among these genes, TACC3, a transforming acidic coiled-coil (TACC) family member could be a potentially druggable target given its role in protecting microtubule stability and centrosome integrity and recent identification of small molecule TACC3 inhibitors as an anticancer agent (Akbulut et al., Mol Cancer Ther 2020).

When comparing differentially expressed pathways in bulk pre-treatment cells of non-responders (post-treatment bone marrow blasts >5%) vs responders (post-treatment bone marrow blasts >5%), we found upregulation of MYC targets, MTORC1 signaling, E2F targets and G2M checkpoint. We also found downregulation of immune-related pathways, namely allograft rejection and interferon-g response. This indicates the possibility of similar transcriptional mechanisms which lead to resistance and immune evasion in LSCs and in bulk populations resistant to therapy.

Finally, we analyzed expression of immunotherapy targets with ongoing clinical trials in AML and compared expression of these targets to CD34+ cells from healthy donors as reference (Figure 2). We found differential expression of targets such as CD123, with high expression in bulk and leukemia subpopulations regardless of genotype, therefore implying the possibility of universally targeting AML with a relatively high therapeutic index. However, targets such as FLT3 or KIT had similar expression in AML compared with bone marrow cells from healthy donors therefore indicating the possibility of affecting normal hematopoiesis in therapies against these targets. Targets such as LAG3 and TIM3 were preferentially expressed in samples with FLT3mt.

Conclusion: In summary, we show characteristic differences in gene expression associated with TP53mt LSCs and identify novel targets and potential mechanisms of resistance. We demonstrate pervasive downregulation of immune related pathways in LSCs or resistant leukemia. These results could impact future therapeutic strategies for these resistant leukemia subsets.

Issa:Novartis, Kura Oncology, Nuprobe: Consultancy; Celgene, Kura Oncology, Syndax, Merck, Cullinan and Novartis: Research Funding. Kanagal-Shamanna:Amgen: Consultancy; Novartis: Consultancy; Aptitude Health: Speakers Bureau; Physicians Education Resource: Speakers Bureau. Daver:Agios, Celgene, SOBI and STAR Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Kartos and Jazz Pharmaceuticals: Other: Data monitoring committee member; 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. Konopleva:AbbVie, Genentech, F. Hoffman La-Roche, Stemline Therapeutics, Amgen, Forty-Seven, Kisoji; Janssen: Consultancy; AbbVie, Genentech, F. Hoffman La-Roche, Eli Lilly, Cellectis, Calithera, Ablynx, Stemline Therapeutics, Agios, Ascentage, Astra Zeneca; Rafael Pharmaceutical; Sanofi, Forty-Seven: Research Funding; Stocks, Reata Pharmaceuticals: Current equity holder in publicly-traded company; Reata Pharmaceuticals, Novartis and Eli Lilly: Patents & Royalties; Forty-Seven; F. Hoffman LaRoche: Honoraria; Stemline Therapeutics, F. Hoffman La-Roche; Janssen: Membership on an entity's Board of Directors or advisory committees. Andreeff:AstraZeneca: Research Funding; Syndax: Consultancy, Research Funding; Pinot Bio: Research Funding; Brooklyn ITX: Research Funding; Medicxi: Consultancy; Breast Cancer Research Foundation: Research Funding; Glycomimetics: Consultancy; NCI: Membership on an entity's Board of Directors or advisory committees; Cancer UK: Membership on an entity's Board of Directors or advisory committees; Leukemia & Lymphoma Society: Membership on an entity's Board of Directors or advisory committees; German Research Council: Membership on an entity's Board of Directors or advisory committees; Kintor Pharmaceutical: Research Funding; Daiichi-Sankyo Inc.: Consultancy, Research Funding; Chimerix: Current holder of stock options in a privately-held company; Reata: Current holder of stock options in a privately-held company; Oxford Biomedical UK: Research Funding; Senti Bio: Consultancy, Research Funding; Aptose: Consultancy, Membership on an entity's Board of Directors or advisory committees; CLL Foundation: Membership on an entity's Board of Directors or advisory committees; Oncolyze: Current holder of stock options in a privately-held company.