Dependency of Spliceosomal Mutant MDS on Innate Immune Signaling
Authors: Lee Stanley C , North Khrystyna , Chen Chi-Chao , Kim Eunhee , Lu Sydney X. , Ki Michelle , Garippa Ralph , Lowe Scott W , Bradley Robert K. , Abdel-Wahab Omar I 
Published: October 2019

Abstract
Mutations in genes encoding RNA splicing factors constitute the most common class of genetic alterations in patients with myelodysplastic syndromes (MDS). These occur as heterozygous point mutations at specific amino acid residues in SF3B1, SRSF2, and U2AF1, and are almost always mutually exclusive with one another. Recent studies have identified that mutations in each of these genes results in activation of the innate immune signaling through altered splicing of mRNAs encoding key enzymes in this pathway. Now, through an unbiased genetic screen as well as focused genetic studies, we have identified that SF3B1-mutant MDS depends on aberrant immune signaling for cell survival.
Recent work has identified that aberrant splicing of MAP3K7 (also known as TAK1; TGF-b Activating Kinase 1) is pervasive across SF3B1-mutant human and mouse cells and results in reduced MAP3K7 protein expression and increased NF-κB signaling. Consistent with this, Map3k7 haploinsufficiency in myeloid cells is known to cause myeloproliferation, while at the same time, complete loss of Map3k7 is intolerable for hematopoietic cells. We therefore hypothesized that partial inhibition of MAP3K7 might preferentially impact SF3B1-mutant cells. To test this hypothesis, we generated mice with inducible deletion of 1 or 2 copies of Map3k7 (Mx1-cre Map3k7fl/+,Mx1-cre Map3k7fl/fl) alone or in the presence of mutant Sf3b1K700E (Mx1-cre Map3k7fl/+Sf3b1K700E/+,Mx1-cre Map3k7fl/flSf3b1K700E/+), along with all controls (Mx1-cre Sf3b1+/+ Map3k7+/+ (Wildtype; WT) and Mx1-cre Sf3b1K700E/+ mice). We then performed bone marrow transplantation (BMT) to assess the effect of Map3k7 deletion on aberrant hematopoiesis driven by mutant SF3B1. Consistent with prior reports, heterozygous deletion of Map3k7did not affect repopulating potential in BMT assays compared to controls while homozygous deletion of Map3k7 resulted in complete failure of hematopoiesis (Figure A). Interestingly, however, in the presence of Sf3b1K700E mutation, deletion of a single copy of Map3k7 completely rescued the hematopoietic defects characteristic of mutant SF3B1 in both mature and immature cells (Figure B-C). These data suggest that inhibition of residual MAP3K7 function may preferentially target SF3B1-mutant MDS cells.
In parallel to the above studies, we also performed a negative selection RNAi screen to uncover novel genetic dependencies in SF3B1-mutant myeloid neoplasms. We performed pooled lentiviral infection of shRNAs targeting ~2,200 genes encoding proteins which are drug targets ("The Druggable Genome") under the control of a doxycycline-inducible vector in isogenic K562 cells expressing the two most commonly occurring SF3B1 mutations, SF3B1K666N and SF3B1K700E, from the endogenous SF3B1 locus. Two individual clones per SF3B1-mutant line were used to improve the robustness of the screen. On Day 21 following shRNA activation, genes with ≥3 shRNAs depleted in SF3B1-mutant cells while remaining unchanged in parental K562 cells were selected. This identified 101 candidates that are potentially synthetic-lethal with SF3B1 mutation (Figure D). Interestingly, pathway analysis of these potential candidates revealed of genes involved in immune and inflammatory signaling as well as in metabolic processes (Figure E). Further target validation was performed using in vitro competitive growth assay in K562 cells, and another set of SF3B1 isogenic lymphoid leukemia cell lines (NALM-6) expressing the same mutations. This revealed consistent dependency of SF3B1-mutant cells on STAT1, an essential component of the interferon (IFN) signaling pathway (Figure F). Upon exposure to Type-I IFNs, SF3B1-mutant K562 cells showed increased transcriptional response in IFN-responsive genes containing interferon stimulated response elements (ISREs) compared with SF3B1 WT cells (Figure G). These data highlight that SF3B1-mutant cells are hyper-responsive to IFN signaling and require intact IFN-signaling responses for cell survival.
Taken together, the above studies indicate that sustained IFN signaling as well as activated innate immune signaling downstream of TAK1 are required for the survival of SF3B1-mutant myeloid cells. These results therefore have important therapeutic implications as they suggest that pharmacologic inhibition of STAT1/Type I IFN activation and/or TAK1 may serve as important therapeutic agents for SF3B1-mutant MDS.
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Disclosures
No relevant conflicts of interest to declare.