Exploiting RAS pathway addiction as a therapeutic vulnerability in myeloid sarcoma Free

Myeloid sarcoma is a distinct form of acute myeloid leukemia (AML) which affects approximately 10-15% of patients (pts) with AML. It is characterized by the proliferation of myeloid blasts forming tumor-like lesions in extramedullary sites, with or without bone marrow involvement. Patients with myeloid sarcoma face a poor prognosis, with a high risk of relapse following conventional chemotherapy. Recent advances in AML therapies have largely bypassed these patients, as they are often excluded from clinical trials. Consequently, treatment of myeloid sarcoma remains reliant on cytotoxic chemotherapy, which yields poor response and survival outcomes similar to those of adverse-risk AML. There is an unmet need for the identification of targetable vulnerabilities to design prospective clinical trials for this patient population.

To address this need, we conducted the first comprehensive genomic and transcriptomic profiling of myeloid sarcoma, with comparison to AML. We assembled a large cohort of myeloid sarcoma patients and performed genomic profiling of myeloid sarcoma and associated bone marrow (BM, whole exome sequencing, n=6, targeted mutation panel, n=36) and transcriptomic sequencing, including bulk RNA-Seq (paired myeloid sarcoma and AML BM n=22, myeloid sarcoma n=15 and AML BM n=11), single cell RNA-Seq (myeloid sarcoma n=3, BM from AML patients with (n=8) and without (n=15) myeloid sarcoma and healthy donor BM, n=5), and spatial transcriptomics using the 10x Visium platform (myeloid sarcoma, n=4). Our genomic data revealed a high prevalence of RAS pathway mutations (KRAS, NRAS, PTPN11, CBL) in myeloid sarcoma (43% of pts) compared to 25% in pts with AML without extramedullary involvement. Notably, RAS pathway activation was also validated by immunohistochemistry, demonstrating phospho-ERK expression in pts without these mutations (n=5) and suggesting that oncogenic addiction to RAS signaling may drive myeloid sarcoma development. Furthermore, whole exome sequencing of myeloid sarcoma and paired BM from Tet2-mutant mice that spontaneously develop myeloid sarcoma (n=3) revealed inactivating mutations in Dusp2, a negative regulator of RAS signaling, in all myeloid sarcomas, highlighting the important role of this pathway in disease development.

Transcriptomic analysis demonstrated that myeloid sarcoma development involves profound changes in both malignant cells and their microenvironment, facilitating a transition from a liquid to a solid tumor state which is marked by clonal evolution and phenotypic changes that enable immune escape. We identified site-specific molecular evolution in myeloid sarcoma with upregulation of genes related to epithelial-mesenchymal transition, adhesion, and apical junctions (SNAI2, VCAN, EMP3, SNTB1). Spatial transcriptomics further revealed extensive tissue remodeling in response to myeloid sarcoma, characterized by increased fibroblasts and vascular cells, indicating adaptation to a solid tumor environment. Additionally, we observed alterations in the BM immune microenvironment, including a depletion of cytotoxic CD8⁺ T cells and an increase in T regulatory cells in myeloid sarcoma, as well as upregulation of HLA-II genes in malignant cells (B2M, HLA-DMA/DMB/DRA/DOB), suggesting aberrant regulation of the immune response in the extramedullary site.

To investigate the role of RAS activation in myeloid sarcoma, we lentivirally transduced human CD34⁺ cord blood cells to express myeloid driver mutations SRSF2^P95H, ASXL1^del1900-1922 and a doxycycline (Dox)-induced NRAS^G12D and transplanted them into NSGS mice. Dox-treated mice developed extramedullary tumors in the spleen, liver and lungs, with a latency of 5-6 weeks. However, withdrawal of Dox one week post-transplantation prevented extramedullary tumor development. To explore therapeutic targeting of RAS signaling, we used a Tet2^-/-/Nras^G12D bone marrow transplant model in Rag2^-/-Il2rg^-/- mice, treating them with the pan-RAS inhibitor RMC-7977 starting 5 days after transplant. While vehicle-treated mice developed extramedullary tumors and succumbed within 3 weeks, RMC-7977-treated mice had significantly reduced extramedullary tumor burden and extended overall survival. In summary, our findings underscore the critical role of RAS pathway activation in myeloid sarcoma development and demonstrate that targeting RAS signaling could be a promising therapeutic strategy for affected patients.