H3B-8800, An Orally Available Small-molecule Splicing ... - PubMed

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Abstract

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.

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Conflict of interest statement

COMPETING FINANCIAL INTERESTS

The authors declare competing financial interests: details are available in the online version of the paper.

Figures

Figure 1

Figure 1

H3B-8800 modulates splicing of WT…

Figure 1

H3B-8800 modulates splicing of WT and mutant SF3B1 spliceosomes in vitro and preferentially…

Figure 1 H3B-8800 modulates splicing of WT and mutant SF3B1 spliceosomes in vitro and preferentially kills SF3B1-mutant cells. (a) The chemical structure of H3B-8800. (b) Competitive binding of increasing concentrations of H3B-8800 to SF3b complexes isolated from cells overexpressing WT or mutant SF3B1 in the presence of 1 nM pladienolide B. The y axis represents the percentage change (% response) of specific binding relative to the DMSO control of 0%. Data are represented as mean ± s.d.; n = 3 experimental replicates. (c,d) Quantification of canonical (c) and aberrant (d) junctions using the Ad2-ZDHHC16 pre-mRNA substrate, in which the original Ad2 intron was substituted with intron 9 of ZDHHC16, in an in vitro splicing assay using nuclear extracts from 293FT cells overexpressing SF3B1K700E and treated with increasing concentrations of H3B-8800. The y axis represents the percent response relative to the DMSO control (0%). Data are represented as mean ± s.d.; n = 3 technical replicates. (e) Viability and apoptosis of pancreatic cells bearing WT (Panc10.05, HAPFII, CFPAC1, and Panc04.03 in green) or mutant (Panc05.04 in violet) SF3B1 treated with increasing concentrations of H3B-8800. Data are represented as mean ± s.d.; n = 3 technical replicates. (f,g) Effect of increasing concentrations of H3B-8800 on splicing of MBD4 mature mRNA (open circles) and MBD4 pre-mRNA (black circles) (f) and MAP3K7 aberrant junction expression (open diamonds) (g) in Panc05.04 cells as quantified by TLDA after 6 h of treatment with H3B-8800. Data are represented as mean ± s.e.m.; n = 3 technical replicates. (h) Viability of HCT116 parental cells and clones carrying the pladienolide-resistance mutation SF3B1R1074H or PHF5AY36C. Data are represented as mean± s.d.; n = 3 technical replicates. (i) Binding of U2 snRNA to the canonical branchpoint sequence (2′-O-methyl BBR oligonucleotide in red with the branchpoint adenosine bolded) in nuclear extracts from the same HCT116 cells as in h. Nuclear extracts were incubated in the presence or absence of ATP and H3B-8800; band quantification is shown below each lane. The functional 17S U2 snRNP was formed via ATP-dependent binding of the SF3b complex to the nonfunctional 12S U2 snRNP particle.
Figure 2

Figure 2

H3B-8800 modulates splicing and selectively…

Figure 2

H3B-8800 modulates splicing and selectively kills SF3B1-mutant leukemia cells in vivo . ( …

Figure 2 H3B-8800 modulates splicing and selectively kills SF3B1-mutant leukemia cells in vivo. (a,b) Mean tumor volume in NSG mice subcutaneously implanted with K562 isogenic cells with (b) or without (a) endogenous mutations in SF3B1. Mice were orally treated with vehicle or H3B-8800 daily at the indicated doses. Mean tumor volumes ± s.e.m. are shown; n = 8 mice per group. (c) Splicing of MBD4 mature mRNA (black open circles), MBD4 pre-mRNA (black circles), and MAP3K7 aberrant junction expression (black open diamonds) in K562 SF3B1K700E tumors after a single oral dose of H3B-8800 (8 mg/kg) as quantified by TLDA. The drug concentration in H3B-8800-treated mice was determined in plasma (red open circles) and tumor (red open squares). Data are represented as mean ± s.e.m.; n = 4 mice per group. The H3B-8800 concentration is shown in ng/ml for plasma and ng/g for tumor. (d) Results from flow cytometry analysis of human versus mouse hematopoietic cells (hCD45 cells versus mCD45 cells) in bone marrow (BM), spleen, liver, lung, and peripheral blood (PB) of PDX-bearing mice with (top) or without (bottom) mutation in SF3B1 following 10 d of vehicle or H3B-8800 treatment (8 mg/kg). Cells were gated on DAPI−mTer119− cells, and the percentages of live Ter119–cells are shown. (e) Quantification of human leukemic cells (hCD45), expressed as log2 of the percentage of hCD45+ cells in HRB-8800-treated mice divided by the percentage of hCD45+ cells in vehicle-treated mice, in each tissue compartment in all PDX models treated with vehicle or H3B-8800. Data are represented as mean ± s.d.; n = 3 mice for SF3B1K700E and 4 for SF3B1WT. (f,g) Spleen (f) and liver (g) weight in PDX mice treated with vehicle or H3B-8800 (8 mg/kg). Data are represented as mean ± s.d.; n = 3 mice for SF3B1K700E and 4 for SF3B1WT. (h) Effects of H3B-8800 (8 mg/kg) on mature mRNA and pre-mRNA MBD4 levels (canonical splicing) and aberrant ZDHHC16 splicing in human cells isolated from PDX mice. The cells were isolated 3 h after the final dose of H3B-8800. Data are represented as mean ± s.d.; n = 3 mice per group. All P values were calculated using a two-tailed Student’s t-test.
Figure 3

Figure 3

H3B-8800 demonstrates preferential activity on…

Figure 3

H3B-8800 demonstrates preferential activity on SRSF2-mutant leukemia in PDX mice. ( a )…

Figure 3 H3B-8800 demonstrates preferential activity on SRSF2-mutant leukemia in PDX mice. (a) Schematic of H3B-8800 testing in CMML PDX mice. Mice were treated orally with 8 mg/kg of H3B-8800 or vehicle for 10 d (in 2 intervals of 5 d each, separated by 2 d). Each sample was engrafted into two NSGS recipient mice, which were then treated with vehicle or H3B-8800 once human CD45+ (hCD45+) cells in peripheral blood were >3% of total peripheral blood mononuclear cells or hemoglobin was ≤11 g/dl. The mice were killed 2 h after the last dose and were analyzed. (b,c) The effect of H3B-8800 on the percentage of peripheral blood hCD45+ cells in spliceosome-mutant (b) and WT (c) PDX models engrafted with an individual spliceosome-mutant or WT tumor (patient numbering is shown in Supplementary Table 2). The treatment period is marked in gray. (d,e) The effect of H3B-8800 on spleen (d) and liver (e) weight in spliceosome-mutant and WT PDX models bearing tumors derived from 3 patients per genotype. Data are represented as mean ± s.d.; n = 3 mice per group. (f) Flow cytometric analysis of hCD45 versus mCD45 cells in bone marrow, spleen, liver, lung, and peripheral blood of vehicle- or H3B-8800-treated mice engrafted with an individual spliceosome-mutant or WT tumor (cells were gated on DAPI−mTer119− cells, and the percentages of live Ter119− cells are shown). (g) Quantification of the proportion of human leukemic cells in H3B-8800-versus vehicle-treated mice (expressed as log2 of the percentage of hCD45+ cells in H3B-8800-treated mice divided by the percentage of hCD45+ cells in vehicle-treated mice) in each tissue compartment in all PDX models. Data are represented as mean ± s.d.; n = 3 mice per group. (h) Immunohistochemical stains for hCD45+ cells in bone marrow or spleen from H3B-8800- versus vehicle-treated mice. Scale bars, 1,000 μm (CMML 5 low-power field), 200 μm (CMML 8 low-power field) and 50 μm (high-power field). (i,j) EZH2 aberrant junction formation as assessed through RT-PCR (i), and mature mRNA and pre-mRNA MBD4 levels as well as levels of CEP57 aberrant junction expression (j) in cDNA from hCD45+ cells isolated 3 h following the last dose of vehicle or H3B-8800. Data are represented as expression relative to GAPDH; mean ± s.d.; n = 3 technical replicates. All P values were calculated using a two-tailed Student’s t test.
Figure 4

Figure 4

H3B-8800 inhibits splicing of short,…

Figure 4

H3B-8800 inhibits splicing of short, GC-rich introns and affects splicing of mRNAs encoding…

Figure 4 H3B-8800 inhibits splicing of short, GC-rich introns and affects splicing of mRNAs encoding spliceosome components. (a) Hexbin plot showing relative abundance of introns based on length (y axis) and GC content (x axis) of all introns of expressed genes in K562 cells (background, left) versus introns retained in SF3B1K700E cells after H3B-8800 treatment (13 nM, right). The percentage of the total intron count contained within each hex is indicated by its color. (b) GC content within introns retained after H3B-8800 treatment of K562 SF3B1K700E cells (red; n = 5,788 introns) versus a randomly selected subset of introns from background (black; n = 10,000 introns). The GC content within the exons that followed is also shown. The solid line represents the mean GC content, and the shaded area surrounding the solid line represents the 95% confidence interval of that measurement. (c) The 3′ splice site motif of introns retained in K562 SF3B1K700E cells following H3B-8800 treatment as compared to background. (d) Branchpoint sequence motif (extending from 6 nt upstream of the branchpoint nucleotide to 4 nt downstream of the branchpoint) representing all branchpoints sequenced by Mercer et al. (left) and only those branchpoints within introns retained in in K562 SF3B1K700E cells following H3B-8800 treatment (right). (e) Top, depiction of two Ad2-derived pre-MRNA substrates: Ad2.14, in which the Py-tract contains five cytosines and five thymines, and Ad2.17, which also contains an alteration in the BPS to reduce binding affinity to U2 snRNP. The branchpoint nucleotide is labelled in red, and the nucleotide that was changed between Ad2.14 and Ad2.17 to alter branchpoint strength is bolded. Bottom, the results from in vitro splicing using these two pre-mRNA substrates. The y axis represents the percent response relative to DMSO control (0%). Data are represented as mean ± s.d.; n = 3 technical replicates. (f) Sashimi plot representing the average read density of three technical replicates in introns 3 and 4 of U2AF2 (top) and introns 20, 21, and 22 of RBM10 (below) of RNA-seq samples from K562 SF3B1K700E cells treated with DMSO or H3B-8800. Exons are shown in black, and introns are shown in blue (DMSO treated) or red (H3B-8800 treated). Relative GC content (derived using a 100-bp sliding window) is depicted in a heat map below the reference sequence. (g) The effect of an increasing concentration of H3B-8800 on the level of U2AF2 pre-mRNA (black squares) and mature mRNA (open squares) in K562 SF3B1K700E cells as quantified by qPCR. Data are represented as mean ± s.e.m.; n = 3 technical replicates. (h) Western blot analysis of U2AF2, SRPK1, and MCL1 protein levels following treatment of K562 SF3B1K700E cells with H3B-8800 or E7107 at the indicated concentrations. β-actin and GAPDH were used as loading controls.
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Comment in

  • A Small-Molecule Splicing Modulator Targets Spliceosome-Mutant Cells. [No authors listed] [No authors listed] Cancer Discov. 2018 Apr;8(4):384. doi: 10.1158/2159-8290.CD-RW2018-038. Epub 2018 Mar 2. Cancer Discov. 2018. PMID: 29500297

References

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    1. Wang L et al. SF3B1 and other novel cancer genes in chronic lymphocytic leukemia. N. Engl. J. Med 365, 2497–2506 (2011). - PMC - PubMed
    1. Harbour JW et al. Recurrent mutations at codon 625 of the splicing factor SF3B1 in uveal melanoma. Nat. Genet 45, 133–135 (2013). - PMC - PubMed
    1. Imielinski M et al. Mapping the hallmarks of lung adenocarcinoma with massively parallel sequencing. Cell 150, 1107–1120 (2012). - PMC - PubMed
    1. Bailey P et al. Genomic analyses identify molecular subtypes of pancreatic cancer. Nature 531, 47–52 (2016). - PubMed
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