Cogle, C. R. | Search bioRxiv

bioRxiv2023

Heterogeneous Nuclear Ribonucleoprotein C is an Indispensable Target in Acute Myeloid Leukemia

Refractory disease is the greatest challenge in acute myeloid leukaemia (AML). Previously, we found vascular-associated AML cells as a source for refractory disease. In this study we use a bioassay of AML cells co-cultured on bone marrow-derived endothelial cells (BMECs) to screen 31 million compounds within a mixture-based synthetic combinatorial library followed by deconvolution leading to the identification of a novel polyamine sulfonamide that is selectively toxic to AML. Using three distinct proteomics methods we identified heterogeneous nuclear ribonucleoprotein C (HNRNPC) as the compound target. Depleting HNRNPC reduces AML cell proliferation, viability, colony formation and fails to establish AML in mice. In contrast, no changes in cell proliferation, survival, or colony outgrowth were observed when depleting HNRNPC in normal hematopoietic or stromal cells. Clinical data show that lower HNRNPC expression independently associates with prolonged survival times in 554 adult and pediatric AML patients. We show that HNRNPC regulates DNA replication initiation and the Myc transcriptional program. Using differential isoform expression and differential alternative splicing analyses, we show significant alternative splicing of MAX after HNRNPC depletion, resulting in MAX transcript isoforms that lack Myc-interacting domains, which we term non-activating MAX isoforms. These Myc-interacting domains are necessary for the obligate heterodimerization of Myc and Max and are critical for Myc transcriptional activation. We further analyzed the alternative splicing landscapes of 538 AML patients and 33 specimens from healthy individuals and show substantial alternative splicing of mRNA in AML patients despite the absence of splicing factor gene mutations. Moreover, we found significantly high expression of non-activating MAX isoforms in healthy individuals, while AML patients highly expressed fully functional MAX isoforms, suggesting that MAX splicing may serve an important functional role in AML. Together, these data show HNRNPC as an indispensable splicing repressor in AML that is necessary for supporting deregulated Myc-Max activity and suggest HNRNPC as a potential therapeutic target for AML.

cancer biology↗

bioRxiv2021

DNMT3A harboring leukemia-associated mutations directs sensitivity to DNA damage at replication forks

Mutations in the DNA methyltransferase 3A (DNMT3A) gene are recurrent in de novo acute myeloid leukemia (AML) and are associated with resistance to standard chemotherapy, disease relapse, and poor prognosis, especially in advanced-age patients. Previous gene expression studies in cells with DNMT3A mutations identified deregulation of cell cycle-related signatures implicated in DNA damage response and replication fork integrity, suggesting sensitivity to replication stress. Here we tested whether pharmacologically-induced replication fork stalling creates a therapeutic vulnerability in cells with DNMT3A(R882) mutations. We observed increased sensitivity to nucleoside analogs such as cytarabine in multiple cellular systems expressing mutant DNMT3A, ectopically or endogenously, in vitro and in vivo. Analysis of DNA damage signaling in response to cytarabine revealed persistent intra-S phase checkpoint activation, accompanied by accumulation of DNA damage in the DNMT3A(R882) overexpressing cells, which was only partially resolved after drug removal and carried through mitosis, resulting in micronucleation. Pulse-chase double-labeling experiments with EdU and BrdU after cytarabine wash-out demonstrated that cells with DNMT3A(mut) were able to restart replication but showed a higher rate of fork collapse. Gene expression profiling by RNA-seq identified deregulation of pathways associated with cell cycle progression and p53 activation, as well as metabolism and chromatin. Together, our studies show that cells with DNMT3A mutations have a defect in recovery from replication fork arrest and subsequent accumulation of unresolved DNA damage, which may have therapeutic tractability. These results demonstrate that, in addition to its role in epigenetic control, DNMT3A contributes to preserving genome integrity during DNA replication.

cancer biology↗