Integrative Analysis of Blast Phase Chronic Myelomonocytic Leukemia Reveals Distinct Transformation Phenotypes Predictive of Drug Response

Introduction: Blastic transformation of chronic myelomonocytic leukemia (CMML) is a common and terminal event. Blast phase CMML (BP-CMML) displays clinical and genetic heterogeneity, but its clonal evolution and transcriptional dynamics are poorly understood. Improved stratification would better characterize transformation phenotypes and identify opportunities for personalized therapy.

Methods: Blasts/HSPCs were FACS sorted (SSC^loCD45^wk) from 43 BP-CMML patients and 7 age-matched healthy controls for bulk RNA-sequencing. Blast immunophenotype by flow cytometry was integrated with clinical metadata in an unsupervised Random Forest classifier. Clonal architecture was investigated using the Tapestri DNA+Protein single cell platform and a bespoke pipeline. Drug testing comprised Cell Titer Glo assay of primary BP-CMML cells.

Results: BP-CMML blasts displayed 2090 differentially expressed genes vs control HSPCs. Upregulated genes (n=1424) included the AP-1 transcription factors (JUN, JUNB, JUND), ATF3, and ID1; and included 110 genes from our previous single cell RNA seq of BP-CMML, including CXCL8, HLX, LGALS1 and MKNK1. We sought genes/pathways upregulated in both bulk and single cell datasets, prioritizing by known function and availability of inhibitors. We identified 12 targets, including RAF mitogenic effectors (VEGFA, AKT, PI3K, MTOR), the pERK downstream effector MKNK1, HIF1A, STAT3/5 and NFKB. BP-CMML cells were consistently sensitive to AKT, MKNK1 and HIF1A inhibition, irrespective of genotype or prior therapy, with an additive effect at therapeutically relevant doses for combined AKT/MKNK1 inhibition.

Observed heterogeneity between BP-CMML cases suggested distinct clinicopathological phenotypes. We integrated clinical metadata with blast immunophenotype (59 independent features) for unsupervised learning by Random Forest, then overlaid RNA-seq. This clearly separated 2 groups ("C1/C2"), with distinctive clinical and molecular features (eg C2 enriched for ASXL1 (p=0.004) and RUNX1 (p=0.044) mutations; C1 for NPM1 (p=0.031)), but no difference in survival. C2 displayed an immature, stem-like phenotype (higher expression of CD34, CD117 and enriched HSC transcriptional signature). C1 reflected a more mature myelomonocytic phenotype (higher CD123, CD13, CD135, CD56) with enrichment of monocytic/granulocytic gene signatures (Fig A).

Amongst genes dysregulated in BP-CMML, 1213 and 402 were unique to C1 or C2, respectively. C1 was enriched for myeloid genes (CD14, S100A8/9, TLR4) and related pathways; C2 for ribosomal, mitogenic (MAP3K8) and FOS family. Genie3 highlighted distinct regulatory networks in C1 and C2, driven by myeloid differentiation (SPI1, TFEB, KLF4) and proto-oncogene (JUND, ATF3, ETV6) transcription factors, respectively. Intriguingly, they displayed different/opposing signatures for resistance to azacitidine (C2>C1; p=0.004), venetoclax (C1>C2; p=0.007) and cytarabine (C1>C2; p=0.0003). We validated for C1 absolute and relative (vs C2) ex vivo resistance to venetoclax, daunorubicin and cytarabine, but saw no difference in azacitidine response.

Multiomic single cell genotyping (n=8) revealed patterns of clonal expansion linked to secondary genetic events, with all harboring acquired mutations in RAS pathway or IDH1/2. Concurrent profiling of 45 surface proteins uncovered subtype-specific differences in origin and extent of differentiation block arising from blasts. Dominant genetic clones in C2 were restricted to phenotypically primitive (CD34+117+) cells with little maturation beyond. By contrast, fully evolved clones in C1 were widely distributed from primitive to mature cells, indicating transformation initiating upstream with a 'leaky' differentiation block and continued multilineage contribution of blasts to the pool of maturing cells (Fig B).

Conclusion: Integration of multimodal data reveals distinct CMML transformation phenotypes associated with unique transcriptional states and drug sensitivities, mirroring the refractoriness of BP-CMML to AML therapy. Subgroups share mitogenic gene expression and ex vivo sensitivity to AKT/MKNK1 inhibition as a promising novel combination therapy. Finally, we describe different routes of clonal evolution, confirming that BP-CMML blasts can retain extensive differentiation capacity to supplant multilevel hematopoiesis after transformation.

Somervaille:Novartis: Consultancy; Abbvie: Consultancy; Imago Biosciences: Research Funding; Oryzon Genomics: Consultancy; CellCentric Ltd: Research Funding; Bristol Myers Squibb: Consultancy. Wiseman:Jazz Pharmaceuticals: Honoraria; Astex Pharmaceuticals: Research Funding; Gilead Sciences: Membership on an entity's Board of Directors or advisory committees; Blueprint Oncology: Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees.