A Novel Ptpn11^E76K/Npm1^cA Murine Model of AML Identifies a Unique Immune Population Relevant to Human Disease

Acute myeloid leukemia (AML) is the most common diagnosed and lethal acute adult leukemia. Traditionally, AML patients were treated with intensive induction chemotherapy ± allogeneic stem cell transplant, which only cures a small subset of individuals. The recent development of targeted therapies directed at driving mutations have improved results, sometimes even in the absence of chemotherapy or allogeneic stem cell transplant. A better understanding of resistance mechanisms to both intensive chemotherapy and targeted therapy will potentially improve outcomes. One genomic group identified in AML that has primary and acquired secondary resistance to targeted therapies (i.e., enasidenib, venetoclax, gilteritinib, and entospletinib) are patients with a protein tyrosine phosphatase non-receptor type 11 (PTPN11) mutation. PTPN11 is the gene that encodes for the protein Shp2 and is involved in the regulation of multiple signaling pathways. A previously described Ptpn11^E76K mouse has been shown to develop multi-lineage leukemias, with relatively equal frequency of B-cell, T-cell, and myeloid disease. While PTPN11 mutations can occur in isolation in AML patients, it is more often found in combination with additional recurrent AML mutations such as NPM1. Therefore, in our study, we describe the generation of a novel mouse model to represent the common co-occurrence of PTPN11 mutations with an NPM1 mutation.

We first sought to determine if the conditionally activated Ptpn11^E76K mutation together with the Npm1^cA mutation canpromote the uniform development of myeloid malignancy. The Ptpn11^E76K/Npm1^cA double mutant mice (n=19) have a median survival of 87 days post poly(I:C) induction compared to 168 days in the published Ptpn11^E76K mice without Npm1^cA. Furthermore, the Ptpn11^E76K/Npm1^cA mice consistently develop acute myeloid leukemia based on flow cytometry data showing a consistent expansion of CD11b⁺cells in the peripheral blood (n=22 of 22) and histopathology demonstrating an accumulation of neoplastic cells of myeloid origin in multiple organs (n=5). These mice have an expansion of white blood cells by six weeks post-induction that continues to expand until the mice meet early removal criteria. These mice do not develop anemia but can have reduced platelet counts relative to single mutation control mice (n=8). Splenocytes from the Ptpn11^E76K/Npm1^cA mice can induce a lethal leukemia in NCG recipients with a median survival ranging from 47 to 70 days (n=3). The NCG recipients develop anemia and thrombocytopenia as the Ptpn11^E76K/Npm1^cA blasts expand.

One of the benefits of a genetic model of AML is the ability to monitor changes in the immune system in response to tumor initiation and expansion. To characterize the immunophenotype of the Ptpn11^E76K/Npm1^cA AML, we have developed a 36-color spectral flow cytometry panel. We found an expansion of blasts cells characterized by CD11b⁺ and cKit⁺ expression in the Ptpn11^E76K/Npm1^cA splenocytes, consistent with other genetic models of AML. However, we also identified an additional sub-population of CD11b⁺ cells that express CD11c⁺, B220⁺, and CD317⁺. Based on this immune phenotype, these cells could represent a population of plasmacytoid dendritic cells (pDCs) that are reported to be immunosuppressive and could therefore contribute to leukemia expansion in this model. While most cases of AML with a pDC expansion (pDC-AML) in patients are associated with RUNX1 mutations, early case reports in the literature have identified pDC-AML patients with a PTPN11 mutation but not a RUNX1 mutation.

In conclusion, we have a created a murine model with a Ptpn11 mutation that consistently develops AML and whose splenocytes can initiate a lethal leukemia in immunocompromised mice. These Ptpn11^E76K/Npm1^cA mice have a mixed population of immature myeloid cells and putative atypical pDCs, which can be seen in a subset of PTPN11-mutated patients with AML. This mouse model emphasizes the importance of understanding the molecular heterogeneity of AML. The Ptpn11^E76K/Npm1^cA mice will be used to understand how different clonal populations interact to promote AML development and is a valuable tool for drug development.

Mullaney:Charles River Laboratories: Current Employment. Eisfeld:Karyopharm Therapeutics: Other: Spouse is current company employee. Byrd:AstraZeneca: Consultancy; Novartis: Consultancy, Honoraria; Kura Oncology, Inc: Consultancy; Syndax: Consultancy; Xencor, Inc: Research Funding; Vincerx Pharma: Current equity holder in publicly-traded company; Janssen Pharmaceuticals, Inc.: Consultancy; TG Therapeutics: Honoraria; Pharmacyclics LLC: Honoraria, Research Funding.