Genomic analyses of cancer have identified recurrent point mutations in the RNA splicing factor-encoding genes SF3B1, U2AF1, and SRSF2 that confer an alteration of function. Cancer cells bearing ...these mutations are preferentially dependent on wild-type (WT) spliceosome function, but clinically relevant means to therapeutically target the spliceosome do not currently exist. Here we describe an orally available modulator of the SF3b complex, H3B-8800, which potently and preferentially kills spliceosome-mutant epithelial and hematologic tumor cells. These killing effects of H3B-8800 are due to its direct interaction with the SF3b complex, as evidenced by loss of H3B-8800 activity in drug-resistant cells bearing mutations in genes encoding SF3b components. Although H3B-8800 modulates WT and mutant spliceosome activity, the preferential killing of spliceosome-mutant cells is due to retention of short, GC-rich introns, which are enriched for genes encoding spliceosome components. These data demonstrate the therapeutic potential of splicing modulation in spliceosome-mutant cancers.
Mutations in RNA splicing factors confer an alteration of function and are common in patients with myelodysplastic syndrome (MDS, ~45%), chronic myelomonocytic leukemia (CMML, ~60%), and acute ...myeloid leukemia (AML) derived from these conditions. Recent data suggest that spliceosome-mutant cells are preferentially sensitive to genetic or pharmacologic splicing modulation compared with wildtype (WT) counterparts. Here, we describe the discovery of H3B-8800, a potent and orally bioavailable modulator of the SF3b complex, and demonstrate efficacy in models of spliceosome mutant myeloid malignancies including a novel xenograft system for CMML.
H3B-8800 was identified through a medicinal chemistry approach aimed at identifying compounds with preferential lethality in spliceosome mutant cells. Using a scintillation proximity assay, we demonstrated that H3B-8800 potently binds to SF3b complexes containing either WT or mutant SF3B1 protein. Consistent with this, H3B-8800 showed dose-dependent modulation of splicing in in vitro biochemical splicing assays and cellular pharmacodynamic assays. Selectivity of H3B-8800 for the SF3b complex was confirmed through observing resistance in cells expressing SF3B1R1074H, an SF3B1 mutation previously shown to confer resistance to natural product splicing modulators.
In the above biochemical and cellular assays, H3B-8800 affected splicing similarly regardless of spliceosome genotype. However, preferential inhibition of in vitro cell growth was observed in isogenic AML cells with endogenous knock-in of SF3B1K700E or SRSF2P95H mutations compared to WT counterparts. In animals xenografted with SF3B1K700E knock-in K562 cells, oral H3B-8800 treatment demonstrated dose-dependent splicing modulation and inhibited tumor growth, while no therapeutic impact was seen in WT controls. Similarly, anti-leukemic efficacy and improved survival were observed with H3B-8800 treatment in mice transplanted with Srsf2P95H/MLL-AF9 mouse AML cells, a result not seen in Srsf2 WT/MLL-AF9 counterpart leukemias.
To understand the preferential effects on spliceosome mutant cells, RNA-seq analysis of isogenic K562 cells treated with H3B-8800 was performed. H3B-8800 induced intron retention and exon skipping, however these effects were not global and introns preferentially retained by H3B-8800 were shorter and more GC-rich compared to those unaffected by drug (Figure A). Interestingly, a substantial number of genes experiencing intron retention with H3B-8800 themselves encoded spliceosome components (Figure B). This suggests that the preferential effect of H3B-8800 on spliceosome mutant cells is due to the exquisite dependency of these cells on normal expression of spliceosome proteins.
Next we aimed to understand the therapeutic potential of H3B-8800 in the context of CMML due to the high frequency of SRSF2 mutations and the need for improved outcome in this disorder. To this end, we developed a xenotransplantation model through direct intrafemoral injection of CD34+ cells from CMML patients into "NSGS" mice: a variant of NSG mice that express human IL3, SCF and GM-CSF. We specifically focused on CMML with <10% marrow blasts. Surprisingly, injection of >200,000 CD34+ cells achieved robust engraftment for all patients (n=7) with rapid lethality (median of 39 days). In vivo H3B-8800 administration substantially reduced leukemic burden in spliceosome-mutant but not spliceosome-WT CMML PDX (Figure C). Furthermore, 2.2-fold reductions in immunophenotypically-defined leukemia initiating cells were seen with H3B-8800 versus vehicle treatment in spliceosome-mutant CMML compared with no change in those mice engrafted with spliceosome-WT CMML.
These data identify a novel therapeutic approach with selective lethality in myeloid cells bearing a spliceosome mutation. Despite the essential nature of splicing, CMML/AML cells without a spliceosome mutation were less sensitive to H3B-8800 compared with potent eradication of mutant counterparts. These data demonstrate the therapeutic potential of splicing modulation in spliceosome mutant cancers and H3B-8800 is currently undergoing clinical evaluation in patients with MDS, AML and CMML.
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Notch pathway signaling plays a fundamental role in normal biological processes and is frequently deregulated in many cancers. Although several hypotheses regarding cancer subpopulations most likely ...to respond to therapies targeting the Notch pathway have been proposed, clinical utility of these predictive markers has not been shown. To understand the molecular basis of gamma-secretase inhibitor (GSI) sensitivity in breast cancer, we undertook an unbiased, de novo responder identification study using a novel genetically engineered in vivo breast cancer model. We show that tumors arising from this model are heterogeneous on the levels of gene expression, histopathology, growth rate, expression of Notch pathway markers, and response to GSI treatment. In addition, GSI treatment of this model was associated with inhibition of Hes1 and proliferation markers, indicating that GSI treatment inhibits Notch signaling. We then identified a pretreatment gene expression signature comprising 768 genes that is significantly associated with in vivo GSI efficacy across 99 tumor lines. Pathway analysis showed that the GSI responder signature is enriched for Notch pathway components and inflammation/immune-related genes. These data show the power of this novel in vivo model system for the discovery of biomarkers predictive of response to targeted therapies, and provide a basis for the identification of human breast cancers most likely to be sensitive to GSI treatment.
Bacterial surface polysaccharides are synthesized from lipid-linked precursors at the inner surface of the cytoplasmic membrane before being translocated across the bilayer for envelope assembly. ...Transport of the cell wall precursor lipid II in Escherichia coli requires the broadly conserved and essential multidrug/oligosaccharidyl-lipid/polysaccharide (MOP) exporter superfamily member MurJ. Here, we show that Bacillus subtilis cells lacking all 10 MOP superfamily members are viable with only minor morphological defects, arguing for the existence of an alternate lipid II flippase. To identify this factor, we screened for synthetic lethal partners of MOP family members using transposon sequencing. We discovered that an uncharacterized gene amj (alternate to MurJ; ydaH) and B. subtilis MurJ (murJBs; formerly ytgP) are a synthetic lethal pair. Cells defective for both Amj and MurJBs exhibit cell shape defects and lyse. Furthermore, expression of Amj or MurJBs in E. coli supports lipid II flipping and viability in the absence of E. coli MurJ. Amj is present in a subset of gram-negative and gram-positive bacteria and is the founding member of a novel family of flippases. Finally, we show that Amj is expressed under the control of the cell envelope stress-response transcription factor σ(M) and cells lacking MurJBs increase amj transcription. These findings raise the possibility that antagonists of the canonical MurJ flippase trigger expression of an alternate translocase that can resist inhibition.
Bacterial surface polysaccharides are synthesized from lipid-linked precursors at the inner surface of the cytoplasmic membrane before being translocated across the bilayer for envelope assembly. ...Transport of the cell wall precursor lipid II inEscherichia colirequires the broadly conserved and essential multidrug/oligosaccharidyl-lipid/polysaccharide (MOP) exporter superfamily member MurJ. Here, we show thatBacillus subtiliscells lacking all 10 MOP superfamily members are viable with only minor morphological defects, arguing for the existence of an alternate lipid II flippase. To identify this factor, we screened for synthetic lethal partners of MOP family members using transposon sequencing. We discovered that an uncharacterized geneamj(alternate to MurJ;ydaH) andB. subtilisMurJ(murJBs
; formerlyytgP)are a synthetic lethal pair. Cells defective for both Amj and MurJBsexhibit cell shape defects and lyse. Furthermore, expression of Amj or MurJBsinE. colisupports lipid II flipping and viability in the absence ofE. coliMurJ. Amj is present in a subset of gram-negative and gram-positive bacteria and is the founding member of a novel family of flippases. Finally, we show that Amj is expressed under the control of the cell envelope stress-response transcription factor σMand cells lacking MurJBsincreaseamjtranscription. These findings raise the possibility that antagonists of the canonical MurJ flippase trigger expression of an alternate translocase that can resist inhibition.
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