Kanna, S. | bioRxiv Science

bioRxiv2023

Cellular taxonomy of the preleukemic bone marrow niche of acute myeloid leukemia

Mutations in hematopoietic stem/progenitor cells (HSPCs) can remain dormant within the bone marrow (BM) for decades before leukemia onset. Understanding the mechanisms by which these mutant clones eventually slead to full blown leukemia is of critical importance to develop strategies to eliminate these clones before they achieve their full leukemogenic potential. Recent data suggest that leukemic stem cells (LSCs) induce alterations within BM microenvironment (BMM) favoring LSC growth over normal HSCs. However, the cross talk between preleukemic stem cells (pLSC) and BMM is not completely understood. We hypothesize that pLSC induces critical changes within the BMM that are critical for leukemogenesis. To address this question, we are using our previously developed murine model of AML that highly recapitulates the human disease, develops AML sporadically with a preleukemic phase in which mice display normal white blood counts (WBCs) and absence of blasts in the BM. Thus, this is an excellent model to evaluate changes in the BMM that occurs during progression into AML. Using this model we performed single cell RNA-sequencing on cells from the BMM compared to wild-type (WT) controls. Overall, we defined the transcriptional profiles of pre-leukemic BMM cells and observed decreased percentages of normal BMM cells such as LepR+ mesenchymal stem cells (MSCs) and endothelial cells (ECs), known to regulate normal HSC function. Concomitantly, we found increases in CD55+ fibroblasts and NG2+ pericytes, that might play a more important role in regulation of pre-LSCs. Preleukemic CD55+ fibroblasts had a higher proliferation rate and showed significant down-regulation of several collagen genes known for regulating extra cellular matrix (ECM) including: Col1a1, Col1a2, Col3a1, Col4a1, and Col6a1, suggesting that ECM remodeling occurs in the early stages of leukemogenesis. Importantly, co-culture assays found that pre-leukemic CD55+ BM fibroblasts expanded pre-LSCs significantly over normal HSCs. In conclusion, we have identified distinct changes in the preleukemic BMM and identified a novel CD55+ fibroblast population that is expanded in preleukemic BMM that promote the fitness of pre-LSCs over normal HSCs. STATEMENT OF SIGNIFICANCEWe have identified changes in the BMM landscape that define a preleukemic BM niche which includes the expansion of a novel CD55+ fibroblast population. These data suggest that a distinct preleukemic BM niche exists and preferentially supports LSC survival and expansion over normal HSCs to promote leukemogenesis.

cancer biology↗

bioRxiv2021

A Budding Yeast Model and Screen to Define the Functional Consequences of Oncogenic Histone Missense Mutations

Somatic missense mutations in histone genes turn these essential proteins into oncohistones, which can drive oncogenesis. Understanding how missense mutations alter histone function is challenging in mammals as mutations occur in a single histone gene. For example, described oncohistone mutations predominantly occur in the histone H3.3 gene, despite the human genome encoding 15 H3 genes. To understand how oncogenic histone missense mutations alter histone function, we leveraged the budding yeast model, which contains only two H3 genes, to explore the functional consequences of oncohistones H3K36M, H3G34W, H3G34L, H3G34R, and H3G34V. Analysis of cells that express each of these variants as the sole copy of H3 reveals that H3K36 mutants show different drug sensitivities compared to H3G34 mutants. This finding suggests that changes to proximal amino acids in the H3 N-terminal tail alter distinct biological pathways. We exploited the caffeine sensitive growth of H3K36 mutant cells to perform a high copy suppressor screen. This screen identified genes linked to histone function and transcriptional regulation, including Esa1, a histone H4/H2A acetyltransferase; Tos4, a forkhead-associated domain-containing gene expression regulator; Pho92, an N6-methyladenosine RNA binding protein and Sgv1/Bur1, a cyclin-dependent kinase. We show that the Esa1 lysine acetyltransferase activity is critical for suppression of the caffeine sensitive growth of H3K36R mutant cells while the previously characterized binding interactions of Tos4 and Pho92 are not required for suppression. This screen identifies pathways that could be altered by oncohistone mutations and highlights the value of yeast genetics to identify pathways altered by such mutations.

genetics↗