Mutant U2AF1-expressing cells are sensitive to pharmacological modulation of the spliceosome

Somatic mutations in spliceosome genes are detectable in ∼50% of patients with myelodysplastic syndromes (MDS). We hypothesize that cells harbouring spliceosome gene mutations have increased sensitivity to pharmacological perturbation of the spliceosome. We focus on mutant U2AF1 and utilize sudemycin compounds that modulate pre-mRNA splicing. We find that haematopoietic cells expressing mutant U2AF1(S34F), including primary patient cells, have an increased sensitivity to in vitro sudemycin treatment relative to controls. In vivo sudemycin treatment of U2AF1(S34F) transgenic mice alters splicing and reverts haematopoietic progenitor cell expansion induced by mutant U2AF1 expression. The splicing effects of sudemycin and U2AF1(S34F) can be cumulative in cells exposed to both perturbations—drug and mutation—compared with cells exposed to either alone. These cumulative effects may result in downstream phenotypic consequences in sudemycin-treated mutant cells. Taken together, these data suggest a potential for treating haematological cancers harbouring U2AF1 mutations with pre-mRNA splicing modulators like sudemycins

Knockdown of HSPA9 induces TP53-dependent apoptosis in human hematopoietic progenitor cells.

Myelodysplastic syndromes (MDS) are the most common adult myeloid blood cancers in the US. Patients have increased apoptosis in their bone marrow cells leading to low peripheral blood counts. The full complement of gene mutations that contribute to increased apoptosis in MDS remains unknown. Up to 25% of MDS patients harbor and acquired interstitial deletion on the long arm of chromosome 5 [del(5q)], creating haploinsufficiency for a large set of genes including HSPA9. Knockdown of HSPA9 in primary human CD34+ hematopoietic progenitor cells significantly inhibits growth and increases apoptosis. We show here that HSPA9 knockdown is associated with increased TP53 expression and activity, resulting in increased expression of target genes BAX and p21. HSPA9 protein interacts with TP53 in CD34+ cells and knockdown of HSPA9 increases nuclear TP53 levels, providing a possible mechanism for regulation of TP53 by HSPA9 haploinsufficiency in hematopoietic cells. Concurrent knockdown of TP53 and HSPA9 rescued the increased apoptosis observed in CD34+ cells following knockdown of HSPA9. Reduction of HSPA9 below 50% results in severe inhibition of cell growth, suggesting that del(5q) cells may be preferentially sensitive to further reductions of HSPA9 below 50%, thus providing a genetic vulnerability to del(5q) cells. Treatment of bone marrow cells with MKT-077, an HSPA9 inhibitor, induced apoptosis in a higher percentage of cells from MDS patients with del(5q) compared to non-del(5q) MDS patients and normal donor cells. Collectively, these findings indicate that reduced levels of HSPA9 may contribute to TP53 activation and increased apoptosis observed in del(5q)-associated MDS

Reduction of TP53 inhibits apoptosis induced by HSPA9 knockdown.

<p>(A) Immunoblot of TP53 following knockdown of TP53 by shRNAs in CD34+ cells grown in erythroid culture conditions for 4 days. (B) CD34+ cells grown in erythroid culture conditions were co-transduced with lentiviral constructs carrying an shRNA targeting TP53 with a hygromycin resistance gene (e.g., shGFP, shTP53-3, or shTP53-4) and an shRNA targeting HSPA9 with a puromycin resistance gene (shGFP, sh433, or sh960). Cells were grown in the presence of both hygromycin and puromycin and the fold change in the percentage of Annexin V+ cells was measured by flow cytometry and normalized to the shGFP-hygromycin/shGFP-puromycin transduced cells (n = 3 technical replicates, representative of 2 independent experiments). *p<0.05, **p<0.01,***p<0.001.</p

HSPA9 and TP53 interact in CD34+ cells and HSPA9 knockdown increases nuclear TP53 levels.

<p>(A) Representative immunoblots of TP53 and HSPA9 following immunoprecipitation with anti-TP53 or an IgG control. (B) Representative immunoblots of TP53 from the cytoplasmic and nuclear fractions of cell lysates following knockdown of HSPA9 in CD34+ cells grown in erythroid culture conditions for 5 days.</p

HSPA9 inhibitor MKT-077 reduces cell growth, induces apoptosis, and increases p21 and BAX expression in CD34+ cells.

<p>(A) Immunoblot of HSPA9, p21, BAX and TP53 following MKT-077 treatment of CD34+ cells grown in erythroid culture conditions for 5 days. (B) CD34+ cells were seeded at equal concentrations in erythroid culture conditions, and the total number of cells was counted daily in the presence of MKT-077. The fold change in cell counts was calculated relative to the number of cells on day 1 (n = 3) (0.5 μM versus control, p<0.001; 2 μM versus control, p<0.001). (C) Quantification of Annexin V+ cells (a surrogate for apoptosis) following treatment of CD34+ cells with various concentrations of MKT-077 for 5 days (n = 3). ***p<0.001.</p

Knockdown of HSPA9 increases TP53 expression and activity in CD34+ cells.

<p>(A) Representative immunoblot of HSPA9 and TP53 protein in CD34+ cells transduced with shRNAs targeting a control gene (GFP) or HSPA9 after 7 days in erythroid culture conditions. (B) Transduced cells were seeded at equal concentrations in erythroid culture media and the total number of cells was counted daily in the presence of puromycin selection. The fold change in cell counts was calculated relative to the number of cells on day 1 (n = 3 independent replicates for each shRNA). (C) Quantification of Annexin V+ cells (a surrogate for apoptosis) following knockdown of HSPA9 in CD34+ cells (n = 3 for each shRNA). (D) Gene Set Enrichment Analysis (GSEA) enrichment score plot for 26 annotated TP53-induced genes. Individual genes in the gene set are represented by a black vertical bar in the middle of the plot. (E) TP53 luciferase reporter assay showed a dose-dependent increase in TP53 activity following HSPA9 knockdown (n = 3). LUC, luciferase. * p<0.05, ** p<0.01, *** p<0.001.</p

Knockdown of HSPA9 increases expression of the TP53 target genes p21 and BAX in CD34+ cells.

<p>(A) Representative immunoblot of p21 and BAX following knockdown of HSPA9 in CD34+ cells. (B) Gene Set Enrichment Analysis (GSEA) enrichment score plot for 13 annotated p21-inhibited genes. Individual genes in the gene set are represented by a black vertical bar in the middle of the plot.</p

Del(5q)-associated MDS bone marrow cells are sensitive to MKT-077 treatment.

<p>(A) Bone marrow (BM) cells from a healthy donor (normal BM) and MDS patients without and with del(5q) (n = 1 each) were treated with various concentrations of MKT-077 for 4 days. The fold change in the percentage of Annexin V+ cells was measured by flow cytometry and normalized to 0 μM of MKT-077 (n = 3, technical replicates). (B) Model of the mechanism of TP53 activation following HSPA9 knockdown. HSPA9 interacts with TP53 in the cytoplasm. HSPA9 reduction leads to the release of TP53 from the HSPA9-TP53 cytoplasmic complex. Translocation of TP53 into the nucleus activates downstream target genes <i>BAX</i> and <i>p21</i>. *p<0.05, **p<0.01, ***p<0.001.</p