Mezigdomide in combination with VTP-50469 demonstrates superior synergy and increased induction of apoptosis in MEN1 wild-type and mutant models. (A-B) Heat maps for proliferation relative to DMSO (top) and ZIP synergy scores (bottom) in the OCI-AML2 and MOLM-13 cell lines after 3 days of treatment. Cells were treated with an 8-point VTP dose curve and a 5-point IMiD or CELMoD dose curve, a subset of which is shown. Data pooled from 3 independent experiments; each with n ≥ 3 technical replicates; each square represents median value from n ≥ 9 data points. ZIP synergy scores ≥10 constitute synergy. (C-D) Apoptosis assessment after 6 days of treatment in OCI-AML2 and MOLM-13 cell lines with percentage of cells staining positive for annexin and DAPI or propidium iodide (PI) by flow cytometry. Data depicted pooled from ≥3 independent experiments, each of which had 4 technical replicates for n ≥ 12 per condition; bars represent mean + SD. Statistical analysis with 2-way ANOVA with Tukey multiple comparisons test. Significant (P < .05) differences between IMiDs/CELMoDs at a given VTP-50469 dose shown; ∗P < .05, ∗∗P < .01, ∗∗∗P < .001, ∗∗∗∗P < .0001. (E) Apoptosis assessment after 5 days of treatment in CD34+-selected human cord blood cells that were KMT2A-AF9 transformed vs untransformed. Data depicted pooled from 3 independent experiments without technical replicates, for n = 3 per condition, bars represent mean + SD. Two-way ANOVA with Sidak multiple comparisons testing for differences between untransformed and KMT2A-AF9 transformed at each drug condition listed. (F-G) MV4;11 cell lines with MEN1 mutations inserted into endogenous MEN1 locus. (F) Heat maps for luminescence relative to DMSO in CellTiter-Glo assays (top) and ZIP synergy scores (bottom). Cells were treated with a 5-point VTP-50469 dose-response curve and 3-point mezigdomide dose response. Data pooled from 3 independent experiments, each with n ≥ 3 technical replicates; each square represents median value from n ≥ 9 data points. (G) Apoptosis assessment after 6 days of treatment with percentage of cells staining positive for annexin and DAPI or PI by flow cytometry. Data depicted pooled from ≥3 independent experiments, each of which had ≥3 technical replicates for n ≥ 11 per condition; bars represent mean + SD. Statistical analysis with 2-way ANOVA with Sidak multiple comparisons test for difference between MEZI 10 nM vs DMSO at each VTP-50469 concentration listed. ∗∗∗P < .001; ∗∗∗∗P < .0001. ns, not significant.

Mezigdomide in combination with VTP-50469 demonstrates superior synergy and increased induction of apoptosis in MEN1 wild-type and mutant models. (A-B) Heat maps for proliferation relative to DMSO (top) and ZIP synergy scores (bottom) in the OCI-AML2 and MOLM-13 cell lines after 3 days of treatment. Cells were treated with an 8-point VTP dose curve and a 5-point IMiD or CELMoD dose curve, a subset of which is shown. Data pooled from 3 independent experiments; each with n ≥ 3 technical replicates; each square represents median value from n ≥ 9 data points. ZIP synergy scores ≥10 constitute synergy. (C-D) Apoptosis assessment after 6 days of treatment in OCI-AML2 and MOLM-13 cell lines with percentage of cells staining positive for annexin and DAPI or propidium iodide (PI) by flow cytometry. Data depicted pooled from ≥3 independent experiments, each of which had 4 technical replicates for n ≥ 12 per condition; bars represent mean + SD. Statistical analysis with 2-way ANOVA with Tukey multiple comparisons test. Significant (P < .05) differences between IMiDs/CELMoDs at a given VTP-50469 dose shown; ∗P < .05, ∗∗P < .01, ∗∗∗P < .001, ∗∗∗∗P < .0001. (E) Apoptosis assessment after 5 days of treatment in CD34+-selected human cord blood cells that were KMT2A-AF9 transformed vs untransformed. Data depicted pooled from 3 independent experiments without technical replicates, for n = 3 per condition, bars represent mean + SD. Two-way ANOVA with Sidak multiple comparisons testing for differences between untransformed and KMT2A-AF9 transformed at each drug condition listed. (F-G) MV4;11 cell lines with MEN1 mutations inserted into endogenous MEN1 locus. (F) Heat maps for luminescence relative to DMSO in CellTiter-Glo assays (top) and ZIP synergy scores (bottom). Cells were treated with a 5-point VTP-50469 dose-response curve and 3-point mezigdomide dose response. Data pooled from 3 independent experiments, each with n ≥ 3 technical replicates; each square represents median value from n ≥ 9 data points. (G) Apoptosis assessment after 6 days of treatment with percentage of cells staining positive for annexin and DAPI or PI by flow cytometry. Data depicted pooled from ≥3 independent experiments, each of which had ≥3 technical replicates for n ≥ 11 per condition; bars represent mean + SD. Statistical analysis with 2-way ANOVA with Sidak multiple comparisons test for difference between MEZI 10 nM vs DMSO at each VTP-50469 concentration listed. ∗∗∗P < .001; ∗∗∗∗P < .0001. ns, not significant.

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