![JNJ-75276617 inhibits menin-KMT2A complex association with chromatin, downregulates menin-KMT2A target genes, and induces myeloid cell differentiation in KMT2A-altered and NPM1-mutant AML cells. (A) A chromatin immunoprecipitation–quantitative polymerase chain reaction (qPCR) assay was performed to assess the binding of menin to target gene promoters (MEIS1, HOXA9, and HOXA10) in response to treatment with JNJ-75276617 for 48 hours. All qPCRs were performed in triplicate. Signals for menin binding in the KMT2A-r MOLM-14 or NPM1c OCI-AML3 samples were normalized to input values, and binding events per 1000 cells were calculated. Compared with dimethyl sulfoxide (DMSO) control, menin binding was decreased in MOLM-14 and OCI-AML3 cells treated with JNJ-75276617 at concentrations from 0.1 to 1.0 μM (MEIS1, 3.2- to 9.8-fold in MOLM-14, 1.4- to 2.6-fold in OCI-AML3; homeobox gene A9 [HOXA9], 2.4- to 3.4-fold in MOLM-14, 14.0- to 24.7-fold in OCI-AML3; homeobox gene A10 [HOXA10], 1.6- to 2.2-fold in MOLM-14, 5.2- to 10.3-fold in OCI-AML3); in all cases, the fold decreases fall outside the 95% confidence interval (CI) of the DMSO control. (B) JNJ-75276617 inhibited expression of menin-KMT2A target genes and increased expression of differentiation genes in KMT2A-altered and NPM1-mutant AML cells. Cells were incubated with the indicated concentrations of JNJ-75276617 for either 48 (MOLM-14) or 72 (OCI-AML3) hours. Relative expression of menin-KMT2A target genes and differentiation markers was calculated by dividing the normalized values of the treated samples by the normalized value of the DMSO control. The experiment was performed 3 times, and the error bars represent the mean ± standard deviation. (C,D) Differential expression of menin-KMT2A target genes and myeloid cell differentiation signature in response to JNJ-75276617 in various leukemia cells. Samples were prepared in duplicate. Microarray analysis was performed on leukemia cell lines in response to JNJ-75276617 treatment for 48 hours. Representative menin-KMT2A PD markers are shown in volcano plots (C). Gene sets involved in myeloid cell differentiation were enriched on treatment of JNJ-75276617 in KMT2A-r (MOLM-14, MV4-11) and NPM1c (OCI-AML3) cell lines as denoted by enrichment score (red), whereas no gene set enrichment was found in KMT2A/NPM1-WT (HL-60, KO-52, and K-562) cells (D). The experiment was performed once.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/144/11/10.1182_blood.2023022480/2/blood_bld-2023-022480-gr2b.jpeg?Expires=1730437983&Signature=0PPiRkwcF~GSUVuyAvar~HZDG4FlXRKk3hN8SHxynDI0t3oLN9nRldNG-5BD3wv~5ILKZz4-0j-3jlV8Mm2WKWE0BwVSIndGrPc3IM1q10B1QTbiK8CspvhOHqp2FG1rPbpSWyt63i37J8MdMdX1HzO7CfqS-auAAhuPUQnlYP33FPSgvPW77KKkK92Pusir577AwMhrWJm0le9YodFpx8Vo~rZ5WnybiAOswyDlU4x30HWtVyug3dgnjdmYj2~CEAzuwnahYhERAIrUG3AdSohBDTjOyOgpyRGte~2rUjYNqPLEZ8w86j2~mVtP93KtGtk3v4-atjWltRT5q0ONPw__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
JNJ-75276617 inhibits menin-KMT2A complex association with chromatin, downregulates menin-KMT2A target genes, and induces myeloid cell differentiation in KMT2A-altered and NPM1-mutant AML cells. (A) A chromatin immunoprecipitation–quantitative polymerase chain reaction (qPCR) assay was performed to assess the binding of menin to target gene promoters (MEIS1, HOXA9, and HOXA10) in response to treatment with JNJ-75276617 for 48 hours. All qPCRs were performed in triplicate. Signals for menin binding in the KMT2A-r MOLM-14 or NPM1c OCI-AML3 samples were normalized to input values, and binding events per 1000 cells were calculated. Compared with dimethyl sulfoxide (DMSO) control, menin binding was decreased in MOLM-14 and OCI-AML3 cells treated with JNJ-75276617 at concentrations from 0.1 to 1.0 μM (MEIS1, 3.2- to 9.8-fold in MOLM-14, 1.4- to 2.6-fold in OCI-AML3; homeobox gene A9 [HOXA9], 2.4- to 3.4-fold in MOLM-14, 14.0- to 24.7-fold in OCI-AML3; homeobox gene A10 [HOXA10], 1.6- to 2.2-fold in MOLM-14, 5.2- to 10.3-fold in OCI-AML3); in all cases, the fold decreases fall outside the 95% confidence interval (CI) of the DMSO control. (B) JNJ-75276617 inhibited expression of menin-KMT2A target genes and increased expression of differentiation genes in KMT2A-altered and NPM1-mutant AML cells. Cells were incubated with the indicated concentrations of JNJ-75276617 for either 48 (MOLM-14) or 72 (OCI-AML3) hours. Relative expression of menin-KMT2A target genes and differentiation markers was calculated by dividing the normalized values of the treated samples by the normalized value of the DMSO control. The experiment was performed 3 times, and the error bars represent the mean ± standard deviation. (C,D) Differential expression of menin-KMT2A target genes and myeloid cell differentiation signature in response to JNJ-75276617 in various leukemia cells. Samples were prepared in duplicate. Microarray analysis was performed on leukemia cell lines in response to JNJ-75276617 treatment for 48 hours. Representative menin-KMT2A PD markers are shown in volcano plots (C). Gene sets involved in myeloid cell differentiation were enriched on treatment of JNJ-75276617 in KMT2A-r (MOLM-14, MV4-11) and NPM1c (OCI-AML3) cell lines as denoted by enrichment score (red), whereas no gene set enrichment was found in KMT2A/NPM1-WT (HL-60, KO-52, and K-562) cells (D). The experiment was performed once.
