Identification of PTPN11 mutations As Early Events in the Development of Acute Myeloid Leukemia

Transformation of normal hematopoietic stem cells to acute myeloid leukemia (AML) occurs in a predictable manner with the acquisition of select chromosomal rearrangements, recurrent mutations, and often microenvironmental inflammation. Our inferred understanding of mutation acquisition in AML is derived from variant allele frequency (VAF) in bulk high-throughput sequencing data which suggests that clonal, but not transforming, hematopoiesis genes are mutated first followed by a subclone of cells that obtain gain-of-function mutations in cell signaling or phosphatase genes (e.g., FLT3-ITD, N-RAS, K-RAS, and PTPN11). Unlike most of these secondarily acquired gene mutations, PTPN11 mutations can occur as germline mutations in Noonan syndrome that promote inflammatory signaling and predispose patients to myeloid malignancies. Additionally, PTPN11 represents a novel gene associated with primary and secondary resistance to multiple targeted therapies in AML. This association further raises the relevance of this gene in leukemia pathogenesis. With the advancement of single-cell sequencing technology, we can now more accurately determine which mutations co-occur in the same cell. Prior work by our group using samples acquired from the Alliance for Clinical Trials in Oncology, demonstrated that mutations in the N-terminal SH2 domain of PTPN11 are associated with early death, and PTPN11 mutations, regardless of location, mediate resistance to chemotherapy in AML. We also found a wide range of PTPN11 mutation VAFs (0.05-0.53). This finding prompted the phylogenetic analysis of PTPN11 mutations as potential root or branch acquisitions in AML transformation.

To explore this, we FACS sorted blast cells (based on CD33⁺ and/or CD34⁺ expression) and T-cells (based on CD3⁺ expression) from primary AML pretreatment samples (n=6) that had both PTPN11 and NPM1 mutations in bulk sequencing. Using a custom 96-gene amplicon panel on the T-cells, we verified the lack of PTPN11 mutations in the T-cells, which would have suggested germline Noonan syndrome mutations. In the blast cells, using the Tapestri Single-cell DNA AML panel, we found that in PTPN11-mutated (PTPN11+) patients with a VAF < 0.4 (n=3), PTPN11 mutations were subclonal as expected and only occurred in a fraction of the NPM1-mutated (NPM1+) cells. Two of the 3 patients with a PTPN11 mutation VAF > 0.4 had a PTPN11 and an NPM1 mutation in the founding clone, suggesting that PTPN11 mutations can be early events in the development of NPM1+ AML. As the original set of samples were sorted prior to sequencing, we recognize that smaller, rare subclones could be missed in the analysis. Therefore, we ran an additional set of patient samples using a DNA + Protein single-cell multi-omic approach. The Tapestri Single-cell DNA Myeloid Clonal Evolution panel was run to detect mutations and the TotalSeq-D Heme Oncology Cocktail from Biolegend was used to identify cell subsets. In this follow-up set of samples, we had a mix of PTPN11+/NPM1+ (n=1) and PTPN11+ (n=3) samples, which all had a PTPN11 mutation VAF > 0.3. In the PTPN11+/NPM1+ sample, there was a wild-type clone and both the PTPN11 and NPM1 mutations appeared in a subsequent clone prior to the addition of a DNMT3A mutation. This data suggests that PTPN11 and NPM1 mutations can cooperatively promote the development of AML. In all 3 of the PTPN11+ samples without NPM1 mutations, we found a clone that only had a PTPN11 mutation. Interestingly, all 3 of these patients also had monosomy 7 and an aberration in chromosome 3 [monosomy 3, inv(3), or t(3;3)] in at least 97% of metaphases. Both the PTPN11+ and PTPN11+/NPM1+ samples provide preliminary evidence for the hypothesis that PTPN11 mutations can be early events in AML development.

More importantly, our single cell analyses demonstrate that the phylogenetic evolution of PTPN11 mutations may follow different paths in AML development. Given the frequency with which PTPN11 mutations participate in resistance to therapy in AML, it is important to understand how order of the mutations affects disease characteristics and how these mutated proteins interact with each other to develop effective targeted therapies. We are currently using a novel mouse model to examine this question.

Support: U10CA180821, U10CA180882, U24CA196171, R35CA198183; Clinicaltrials.gov Identifier: NCT00048958, NCT00899223, NCT00900224; https://acknowledgments.alliancefound.org

Stone:Gemoab: Consultancy; Innate: Consultancy; GSK: Consultancy; Novartis: Consultancy; OncoNova: Consultancy; Syndax: Consultancy; Apteva: Consultancy; BMS: Consultancy; Jazz: Consultancy; Foghorn Therapeutics: Consultancy; Janssen: Consultancy; Aprea: Consultancy; Kura Oncology: Consultancy; Epizyme: Consultancy; BerGenBio: Consultancy; Elevate Bio: Consultancy; Arog: Consultancy, Research Funding; Astellas: Consultancy; Boston Pharmaceuticals: Consultancy; Actinium: Consultancy; Abbvie: Consultancy, Research Funding; Takeda: Consultancy; Syros: Consultancy; Syntrix: Consultancy. Wang:Takeda: Consultancy, Honoraria, Other: Advisory Board; Daiichi Sankyo: Consultancy, Honoraria, Other: Advisory Board; Stemline Therapeutics: Consultancy, Honoraria, Other: Advisory Board, Speakers Bureau; Kura Oncology: Consultancy, Honoraria, Other: Advisory Board, Steering Committee, Speakers Bureau; Gilead: Consultancy, Honoraria, Other: Advisory Board; Genentech: Consultancy; PTC Therapeutics: Consultancy, Honoraria, Other: Advisory Board; GlaxoSmithKline: Consultancy, Honoraria, Other: Advisory Board; Astellas: Consultancy, Honoraria; Rafael Pharmaceuticals: Other: Data Safety Monitoring Committee; Macrogenics: Consultancy; Dava Oncology: Consultancy, Speakers Bureau; Mana Therapeutics: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria, Other: member of data monitoring committee ; Pfizer: Consultancy, Honoraria, Other: Advisory Board, Speakers Bureau; BMS/Celgene: Membership on an entity's Board of Directors or advisory committees; Kite Pharmaceuticals: Consultancy, Honoraria, Other: Advisory Board; Jazz Pharmaceuticals: Consultancy, Honoraria, Other: Advisory Board; Novartis: Consultancy, Honoraria, Other: Advisory Board. 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. Eisfeld:Karyopharm Therapeutics: Other: Spouse is current company employee. Byrd:Xencor, Inc: Research Funding; Pharmacyclics LLC: Honoraria, Research Funding; Syndax: Consultancy; Novartis: Consultancy, Honoraria; Kura Oncology, Inc: Consultancy; Janssen Pharmaceuticals, Inc.: Consultancy; Vincerx Pharma: Current equity holder in publicly-traded company; TG Therapeutics: Honoraria; AstraZeneca: Consultancy.