KRAS G12D Specific Inhibitors Relieve Erythroid Differentiation Block and Modulate Inflammatory Pathways at the Single Cell Level in Myeloid Malignancies
Authors: Kravets Leah , Saurty-Seerunghen Mirca S. , Verma Divij , Fromowitz Ariel , Sahu Srabani , Rockwell Bradley , Sakaguchi Olivia , Narayanagari Swathi , Gordon Mitchell Shanisha , Carbajal Milagros , Aluri Srinivas , Wang Jingli , Lafave Lindsay , Konopleva Marina , Nam Anna S. , Verma Amit 
Published: December 2024

Introduction:
Myelodysplastic Syndrome (MDS) and Acute Myeloid Leukemia (AML) are characterized by a hematopoietic differentiation block that leads to anemia, a major source of morbidity and mortality. These myeloid malignancies and clonal hematopoiesis (CH) are associated with a myeloid bias and heightened inflammation, but the mechanisms of these phenomenon are not well elucidated. KRAS G12D (KRASG12D) mutation is seen in ~5% of MDS/AML and associated with a poor prognosis, currently with no approved precision medicine strategies. Novel KRASG12D inhibitors are being developed for solid tumors, but no data exists testing their efficacy in myeloid malignancies. Thus, we examined the impact of KRASG12D mutation and KRAS inhibition on human hematopoietic differentiation.
Results:
To first define the impact of KRASG12D on human hematopoiesis, we performed single-cell RNA-seq coupled with mutational calling via Genotyping of Transcriptomics (GoT) on primary KRASG12D samples. GoT of a CH sample with isolated KRASG12D revealed that the KRASG12D mutated (MT) cells were overrepresented in the monocytic compartment. MT cells also displayed an inflammatory signature, including elevated IL-8 expression.
GoT analysis of KRASG12D AML samples (N=3) revealed that the MT cells were enriched in a distinct AML subpopulation, defined by a stem/progenitor-like expression profile, withupregulated RUNX1, ETV6, JARID2, and ZEB2. Additionally, CD83, an immune checkpoint molecule, was elevated in this AML subgroup. Differential gene expression analysis between CD83+ MT vs. CD83+ WT cells revealed overexpression of IL-6, FCN1, CXCL3 in MT cells, suggesting an inflamed phenotype. Consistent with CD83 upregulation in the stem/progenitor-like subgroup, sorted CD83+ cells from KRASG12D primary MDS samples create more colonies in vitro when compared to sorted CD83- cells.
To test the impact of KRAS-targeted therapy on isolated KRASG12D clones (without confounding co-occurring mutations), we treated the KRASG12D CH sample with a clinically valid KRASG12D inhibitor MRTX1133 and a pan-KRAS inhibitor BI-2865. Treatment with both inhibitors decreased total monocytic population while increasing erythrocytes at the single cell level. We validated this finding by treating primary KRASG12D mutant CH, MDS and AML samples (N=3) with both KRAS-inhibitors in clonogenic assays. Inhibition of KRASG12D led to increased erythroid colonies, associated with increased Glycophorin A and CD71 expressionby flow cytometry. This demonstrates that KRAS-inhibition may rescue erythroid differentiation block.
As we observed that KRASG12D induced inflammatory signaling, we hypothesized that KRAS inhibition may rescue erythroid differentiation by suppressing inflammation. Consistently, the KRASG12D-induced inflammatory signature, including IL-8, was downregulated upon treatment with MRTX1133 and BI-2865. We therefore tested whether IL-8 antibody treatment may rescue erythroid differentiation. Indeed, IL-8 antibody in vitro treatment led to increased Glycophorin A and CD71 expression, suggesting that KRAS inhibition may rescue KRASG12D -mediated anemia via downregulation of IL-8-mediated inflammation.
Conclusion:
These data demonstrate that KRASG12D induces a monocytic differentiation bias associated with inflammation in CH. In AML, KRAS mutant cells are enriched in a novel stem progenitor-like inflamed population, characterized by CD83 positivity. We demonstrate the efficacy of novel, clinically relevant KRASG12D inhibitors in relieving IL8-mediated inflammation and erythroid differentiation block in KRASG12D neoplasms. These data support investigations of KRASG12D inhibitors in myeloid malignancy clinical trials. Ongoing studies test the efficacy of in vivo targeting of KRASG12D clones in murine and xenograft models.