Distinct CMML Transformation Phenotypes Associated With Drug Sensitivity

Kristian Gurashi

In new data presented during the American Society of Hematology 2022 Annual Meeting, investigators found unique characteristics for patients with chronic myelomonocytic leukemia (CMML).

A team, led by Kristian Gurashi, Division of Cancer Sciences, The University of Manchester, sought an improved stratification would better characterize transformation phenotypes and identify opportunities for personalized therapy.

The Disease

Blastic transformation of CMML can be common and terminal for patients.

However, blast phase CMML (BP-CMML) has shown clinical and genetic heterogeneity, while its clonal evolution and transcriptional dynamics are poorly understood.

In the study, the investigators sorted blasts/HSPCs from 43 patients with BP-CMML, as well as 7 age-matched healthy controls for bulk RNA-sequencing. They then integrated blast immunophenotype by flow cytometry with clinical metadata in an unsupervised Random Forest classifier and investigated clonal architecture using the Tapestri DNA+Protein single cell platform and a bespoke pipeline.

Drug testing comprised Cell Titer Glo assay of primary BP-CMML cells.

Different Genes

The investigators identified 2090 differentially expressed genes through BP-CMML blasts compared to 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.

The team looked for genes and pathways upregulated in both bulk and single cell datasets and prioritized them by known function and availability of inhibitors.

Overall, there were 12 targets identified, 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, regardless of genotype or prior therapy, with an additive effect at therapeutically relevant doses for combined AKT/MKNK1 inhibition.

Heterogeneity

The observed heterogeneity between BP-CMML cases suggested distinct clinicopathological phenotypes and the investigators integrated clinical metadata with blast immunophenotype with 59 independent features for unsupervised learning by Random Forest, then overlaid RNA-seq.

This clearly separated 2 groups with distinctive clinical and molecular features (P = 0.004) and RUNX1 (P = 0.044) mutations; C1 for NPM1 (P = 0.031).
However, there was no difference in survival. C2 showed an immature, stem-like phenotype with a higher expression of CD34, CD117 and enriched HSC transcriptional signature. On the other hand, C1 reflected a more mature myelomonocytic phenotype with higher CD123, CD13, CD135, CD56 and enrichment of monocytic/granulocytic gene signatures.

For the genes dysregulated in BP-CMML, 1213 and 402 were unique to C1 or C2, respectively and C1 was enriched for myeloid genes (CD14, S100A8/9, TLR4) and related pathways; C2 for ribosomal, mitogenic (MAP3K8) and FOS family.

Genie3 had distinct regulatory networks in C1 and C2, driven by myeloid differentiation (SPI1, TFEB, KLF4) and proto-oncogene (JUND, ATF3, ETV6) transcription factors, respectively. These genes 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).

The investigators validated for C1 absolute and relative compared to C2 ex vivo resistance to venetoclax, daunorubicin and cytarabine, but saw no difference in azacitidine response.

Multiomic single cell genotyping (n = 8) resulted in 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 and 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.

“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,” the authors wrote. “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.”

The study, “Integrative Analysis of Blast Phase Chronic Myelomonocytic Leukemia Reveals Distinct Transformation Phenotypes Predictive of Drug Response,” was published online by ASH 2022.