ASXL1 mutations are associated with a response to alvocidib and 5-azacytidine combination in myelodysplastic neoplasms

Abstract

Inhibitors of anti-apoptotic BCL-2 family proteins in combination with chemotherapy and hypomethylating agents (HMA) are promising therapeutic approaches in acute myeloid leukemia (AML) and high-risk myelodysplastic syndromes (MDS). Alvocidib, a cyclin-dependent kinase 9 (CDK9) inhibitor and indirect transcriptional repressor of the anti-apoptotic factor MCL-1, has previously shown clinical activity in AML. Availability of biomarkers for response to the alvocidib + 5-azacytidine (5-AZA) could also extend the rationale of this treatment concept to high-risk MDS. In this study, we performed a comprehensive in vitro assessment of alvocidib and 5-AZA effects in N=45 high-risk MDS patients. Our data revealed additive cytotoxic effects of the combination treatment. Mutational profiling of MDS samples identified ASXL1 mutations as predictors of response. Further, increased response rates were associated with higher gene expression of the pro-apoptotic factor NOXA in ASXL1-mutated samples. The higher sensitivity of ASXL1 mutant cells to the combination treatment was confirmed in vivo in ASXL1Y588X transgenic mice. Overall, our study demonstrated augmented activity for the alvocidib + 5-AZA combination in higher-risk MDS and identified ASXL1 mutations as a biomarker of response for potential stratification studies.

Figure 1.

The combination of alvocidib and 5-AZA exhibited an additive effect on the cell viability in human high-risk myelodysplastic syndromes samples. (A) Dose response curves were generated after alvocidib (Alvo) treatment for 24 hours (h) (left panel) and 5-azacytidine (5-AZA) treatment for 48 h (right panel) for N=16 myelodysplastic syndromes (MDS) patients and N=12 hematologically healthy controls (HC). Cell viability was measured using CTG assay. (B) Cell viability (CTG assay) was measured in N=45 MDS patient samples based on mean cell viability (IC30) concentrations of both drugs; median ± interquartile range (IQR), Friedman test with Dunn’s multiple comparisons. (C) The percentages of apoptotic cells (left panel) and apoptotic + dead cells (right panel) were measured using Annexin V assay in N=24 MDS patient samples; median ± IQR; Friedman test with Dunn’s multiple comparisons. (D) Cell viability (CTG assay, left panel) and percentages of apoptotic + dead cells (right panel) were compared after alvocidib + 5-AZA treatment in N=45 MDS patients versus N=11 HC; median ± IQR, Mann-Whitney U test. HC sample with DNMT3A W305fs frameshift mutation is labeled in dark red color. (E) MDS-L xenotransplantation experimental design and fold change in MDS-L expansion in blood between day 50 and day 21 of treatment of NSGS mice injected with MDS-L cells. Expansion fold change = % MDS-L cells at day 50 / % MDS-L cells at day 21. Expansion fold changes are presented as log10 transformed values. Box plots show medians ± IQR; whiskers indicate minimum and maximum; Kruskal-Wallis test with Dunn’s multiple comparisons. TX: xenotransplantation; DMSO: dimethyl sulfoxide.

Figure 2.

Mutational profiles in alvocidib and alvocidib + 5-AZA treated samples. (A) Bone marrow (BM) mononuclear cells (MNC) of myelodysplastic syndromes (MDS) patients (N=40) were subjected to myeloid panel deep sequencing. The mutational data for other N=5 MDS samples were obtained from the medical records. The mutations in MDS-associated genes were found in N=44 patients and presented as oncoplot. (B, C) MDS patients (N=44) were distributed into responder and non-responder groups based on the mean cell viability after alvocidib treatment (B) and alvocidib + 5-azacytidine (5-AZA) treatment (C) in CTG assay. Individual mutations for responders versus non-responders are presented as co-oncoplots. Del: deletion; Ins: insertion.

Figure 3.

ASXL1 mutations are associated with increased sensitivity to alvocidib and alvocidib + 5-AZA treatment. (A-C) The association of cell viability (IC30) in CTG assay with the presence of specific mutations for N=45 myelodysplastic syndromes (MDS) samples; median ± interquartile range (IQR), Mann-Whitney U test. (D) Dose response curves after 24 hours of alvocidib treatment were generated for N=11 ASXL1 wild-type (WT) and N=5 ASXL1 mutant (mut) patient samples (CTG assay).

Figure 4.

Asxl1 mutation sensitizes myelodysplastic syndromes cells to alvocidib and alvocidib + 5-AZA treatment in a mouse model. (A) Bone marrow (BM) mononuclear cells (MNC) from N=2 wild-type (WT) and N=2 Asxl1^Y588X transgenic (Tg) mice were treated with alvocidib (Alvo), 5-azacytidine (5-AZA) or alvocidib + 5-AZA for 8 hours (h). Cell viability was determined using CTG assay; data are combined for N=2 mice and N=5 replicates for each mouse and presented as mean ± standard deviation (SD); unpaired Student’s t test. (B) Experimental design for in vivo assessment of alvocidib + 5-AZA combination in Asxl1^Y588X Tg mice. (C) The percentages of CD45.1⁺ (healthy recipient mice) and CD45.2⁺ cells (Asxl1^Y588X Tg leukemic donor mice) in the bone marrow (BM) were analyzed by flow cytometry after treatment with 5-AZA and alvocidib + 5-AZA. The data for individual mice before and after treatment are shown; Wilcoxon matched-pairs signed rank test. (D) Histological analysis of BM cytospins at the treatment endpoint in recipient CD45.1⁺ mice; median ± interquartile range; Kruskal-Wallis test with Dunn’s multiple comparisons. Arrows on the histological images indicate immature blasts. IP: intraperitoneal.

Figure 5.

ASX L1 mutant myelodysplastic syndromes hematopoietic stem and progenitor cells overexpress NOXA. The expression of MCL-1 and NOXA genes was assessed in hematopoietic stem and progenitor cells (HSPC) of ASXL1 wild-type (WT) and ASXL1-mutated (mut) patients using real-time quantitative polymerase chain reaction; median ± interquartile range, Mann-Whitney U test. Outlier values were removed using GraphPad Prism 8.4.3 software (ROUT method with Q factor =1%).