The Serine/Threonine Kinase HASPIN Is a Novel Dependency in t(8;21) AML

Background: t(8;21) Acute Myeloid Leukemia (AML) is a common subtype of AML. Patients of t(8;21) AML are relatively young (median age of 30s) compared to all AML patients (median age of late-60s). Although over 50% of t(8;21) AML patients respond better to initial chemotherapy than other AMLs, substantial numbers of them experience disease relapse with few options for follow-up treatment, necessitating an improvement to standards of care. Pan-tyrosine kinase inhibitors have been used to effectively treat several leukemia subtypes, suggesting targeted inhibition strategies are viable when utilized in mono or combination therapy. To extend the utility of this therapeutic strategy, additional drug targets must be found. Here, we present the serine/threonine kinase HASPIN as a novel kinase dependency in t(8;21) AML and as a potential therapeutic target for targeted inhibition therapy.

Aim: To identify novel kinase dependencies that can be used as therapeutic targets in the treatment of t(8;21) AML and to characterize the molecular basis of the dependency.

Results: To identify novel kinase dependencies in t(8;21) AML, we employed a human kinase domain targeted CRISPR screen in two t(8;21) AML cell lines (Kasumi-1 and SKNO-1). HASPIN was identified as one of the top hits that dropped out at levels comparable to positive controls, including ribosomal proteins, RNA/DNA polymerases, cell cycle regulators, and other housekeeping genes. HASPIN dependency was further validated in both t(8;21) AML cell lines using two unique sgRNAs. Importantly, HASPIN has several commercially available inhibitors shown to efficiently suppress various cancer subtypes, but their effects in leukemia remain undescribed. Furthermore, HASPIN knockout mice are fertile, viable, and apparently normal, suggesting HASPIN is dispensable or compensated for in healthy cells in a way not mirrored by cancer cells.

The functional role of HASPIN in AML has not been explored. Therefore, we examined the function of HASPIN by transduction of t(8;21) AML cells with two unique HASPIN targeting shRNAs to induce protein depletion. HASPIN knockdown caused significant reductions in proliferation rate in t(8;21) AML cells (Fig. 1A). Additionally, cell cycle analysis revealed that HASPIN depletion caused an increase of cells in G0-G1 phase with corresponding reductions in S and G2-M phases, suggesting cell cycle arrest.

Mass spectrometry studies suggest that HASPIN interacts with and phosphorylates several important transcription and splicing factors (Maiolica et al., 2014 Molecular and Cellular Proteomics). To investigate whether HASPIN regulates transcription and RNA splicing, we performed deep RNA-seq of t(8;21) AML cells transduced with control or HASPIN shRNAs. Our differential gene expression and GSEA analyses of HASPIN knockdown t(8;21) AML cells found negative enrichments for terms related to cell cycle regulation and DNA repair and positive enrichment for terms related to anti-leukemia phenotypes. Importantly, alternative splicing analysis revealed significant differential RNA splicing events in response to HASPIN knockdown. These results support initial hypotheses regarding HASPIN's functional roles in transcription and RNA splicing and hint at the mechanistic underpinnings of HASPIN dependency in t(8;21) AML.

Finally, to assess therapeutic potential of HASPIN inhibition, t(8;21) AML cells and healthy human CD34+ hematopoietic progenitor cells were treated with titrated HASPIN inhibitor, CHR-6494. Strikingly, t(8;21) AML cells were found to be nearly 15-fold more sensitive to HASPIN inhibition than normal hematopoietic progenitors by IC50 (Fig. 1B).

Summary: We have identified HASPIN, a novel kinase dependency in t(8;21) AML cell lines. HASPIN depletion significantly reduces growth rate of t(8;21) AML cells and causes cell cycle arrest into G0-G1 phase. RNA-seq analysis suggests HASPIN knockdown dysregulates cell cycle checkpoint, DNA repair, and leukemia phenotype genes in t(8;21) AML cells. As a potential RNA splicing regulator, HASPIN knockdown induces significant changes in alternative splicing events. Finally, t(8;21) AML cells are significantly more susceptible to HASPIN inhibition than healthy hematopoietic progenitors, demonstrating HASPIN as a potentially promising therapeutic target for chemo resistant t(8;21) AML patients.

Zhang:CurePharma: Current Employment, Other: Founder.