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Clec12a expression is regulated by NUP98::NSD1. (A) CLEC12A mRNA expression among different cytogenetic AML subgroups, derived from the Leucegene cohort (NUP98::NSD1, n = 7; CN-AML, n = 168; N/K-RASmut, n = 87; N/K-RASwt, n = 350; complex KT, n = 139; CBFB::MYH11, n = 29; CEBPAmut, n = 16; MLL:MLLT3, n = 42; PML::RARA, n = 15; RUNX1::RUNX1T1, n = 21; normal CD34 + , n = 17). Values are in log (FPKM). (B) CLEC12A mRNA expression among different AML KTs and mutations, derived from the Balgobind et al cohort ( GSE17855 ; NUP98::NSD1, n = 11; CN-AML, n = 39; MLL::MLT3, n = 47; N/K-RASmut, n = 41; N/K-RASwt, n = 196; CEBPAmut, n = 16; cKITmut, n = 18; FLT3-ITD, n = 48; CBFB::MYH11, n = 27; PML::RARA, n = 19; RUX1::RUNX1T1, n = 28; other, n = 45; unknown, n = 25; NPM1mut, n = 17). Values are in normalized microarray intensity (RMA). (C) Reverse <t>transcriptase</t> <t>PCR</t> revealing Clec12a mRNA expression in murine BM cells transduced with either NRASG12D, NUP98::NSD1, or NUP98::NSD1+NRASG12D (n = 3; mean ± standard error of the mean [SEM]). (D) Flow cytometric representation of Clec12a protein expression in murine hematopoietic BM cells transduced with NUP98::NSD1+NRASG12D. Cells were gated on GFP + BFP + . (E) Normalized Clec12a expression for NUP98 CTL and NUP98::NSD1 murine-expressing cells. Clec12a is highly expressed in NUP98::NSD1-expressing cells (off Dox) compared with NUP98 CTL cells and is strongly downregulated if NUP98-NSD1 expression is shut off (day 3_on_Dox and day 5_on_Dox). Data are presented as boxplots in which the center line represents the median, the boundaries of the box indicate the first (25th percentile) and third (75th percentile) quartiles, and the whiskers extend to 1.5× interquartile range from the hinges. Data points beyond this range are revealed as individual dots and represent outliers. (F) Publicly available NUP98::NSD1 chromatin immunoprecipitation-sequencing data reveal binding of NUP98::NSD1 to regions with proximal or distal enhancer-like signatures according to the ENCODE candidate cis-Regulatory Elements atlas ( GSE112928 ). CTL, control; Dox, doxycycline; FPKM, fragments per kilobase per million; H3K4me3, histone 3, lysine 4 trimethylation; KT, karyotype; mRNA, messenger RNA; RMA, robust multi-array analysis.
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Clec12a expression is regulated by NUP98::NSD1. (A) CLEC12A mRNA expression among different cytogenetic AML subgroups, derived from the Leucegene cohort (NUP98::NSD1, n = 7; CN-AML, n = 168; N/K-RASmut, n = 87; N/K-RASwt, n = 350; complex KT, n = 139; CBFB::MYH11, n = 29; CEBPAmut, n = 16; MLL:MLLT3, n = 42; PML::RARA, n = 15; RUNX1::RUNX1T1, n = 21; normal CD34 + , n = 17). Values are in log (FPKM). (B) CLEC12A mRNA expression among different AML KTs and mutations, derived from the Balgobind et al cohort ( GSE17855 ; NUP98::NSD1, n = 11; CN-AML, n = 39; MLL::MLT3, n = 47; N/K-RASmut, n = 41; N/K-RASwt, n = 196; CEBPAmut, n = 16; cKITmut, n = 18; FLT3-ITD, n = 48; CBFB::MYH11, n = 27; PML::RARA, n = 19; RUX1::RUNX1T1, n = 28; other, n = 45; unknown, n = 25; NPM1mut, n = 17). Values are in normalized microarray intensity (RMA). (C) Reverse <t>transcriptase</t> <t>PCR</t> revealing Clec12a mRNA expression in murine BM cells transduced with either NRASG12D, NUP98::NSD1, or NUP98::NSD1+NRASG12D (n = 3; mean ± standard error of the mean [SEM]). (D) Flow cytometric representation of Clec12a protein expression in murine hematopoietic BM cells transduced with NUP98::NSD1+NRASG12D. Cells were gated on GFP + BFP + . (E) Normalized Clec12a expression for NUP98 CTL and NUP98::NSD1 murine-expressing cells. Clec12a is highly expressed in NUP98::NSD1-expressing cells (off Dox) compared with NUP98 CTL cells and is strongly downregulated if NUP98-NSD1 expression is shut off (day 3_on_Dox and day 5_on_Dox). Data are presented as boxplots in which the center line represents the median, the boundaries of the box indicate the first (25th percentile) and third (75th percentile) quartiles, and the whiskers extend to 1.5× interquartile range from the hinges. Data points beyond this range are revealed as individual dots and represent outliers. (F) Publicly available NUP98::NSD1 chromatin immunoprecipitation-sequencing data reveal binding of NUP98::NSD1 to regions with proximal or distal enhancer-like signatures according to the ENCODE candidate cis-Regulatory Elements atlas ( GSE112928 ). CTL, control; Dox, doxycycline; FPKM, fragments per kilobase per million; H3K4me3, histone 3, lysine 4 trimethylation; KT, karyotype; mRNA, messenger RNA; RMA, robust multi-array analysis.
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reverse transcriptase quantitative polymerase chain reaction rt qpcr rna  (Qiagen)
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Qiagen reverse transcriptase quantitative polymerase chain reaction rt qpcr rna
Clec12a expression is regulated by NUP98::NSD1. (A) CLEC12A mRNA expression among different cytogenetic AML subgroups, derived from the Leucegene cohort (NUP98::NSD1, n = 7; CN-AML, n = 168; N/K-RASmut, n = 87; N/K-RASwt, n = 350; complex KT, n = 139; CBFB::MYH11, n = 29; CEBPAmut, n = 16; MLL:MLLT3, n = 42; PML::RARA, n = 15; RUNX1::RUNX1T1, n = 21; normal CD34 + , n = 17). Values are in log (FPKM). (B) CLEC12A mRNA expression among different AML KTs and mutations, derived from the Balgobind et al cohort ( GSE17855 ; NUP98::NSD1, n = 11; CN-AML, n = 39; MLL::MLT3, n = 47; N/K-RASmut, n = 41; N/K-RASwt, n = 196; CEBPAmut, n = 16; cKITmut, n = 18; FLT3-ITD, n = 48; CBFB::MYH11, n = 27; PML::RARA, n = 19; RUX1::RUNX1T1, n = 28; other, n = 45; unknown, n = 25; NPM1mut, n = 17). Values are in normalized microarray intensity (RMA). (C) Reverse <t>transcriptase</t> <t>PCR</t> revealing Clec12a mRNA expression in murine BM cells transduced with either NRASG12D, NUP98::NSD1, or NUP98::NSD1+NRASG12D (n = 3; mean ± standard error of the mean [SEM]). (D) Flow cytometric representation of Clec12a protein expression in murine hematopoietic BM cells transduced with NUP98::NSD1+NRASG12D. Cells were gated on GFP + BFP + . (E) Normalized Clec12a expression for NUP98 CTL and NUP98::NSD1 murine-expressing cells. Clec12a is highly expressed in NUP98::NSD1-expressing cells (off Dox) compared with NUP98 CTL cells and is strongly downregulated if NUP98-NSD1 expression is shut off (day 3_on_Dox and day 5_on_Dox). Data are presented as boxplots in which the center line represents the median, the boundaries of the box indicate the first (25th percentile) and third (75th percentile) quartiles, and the whiskers extend to 1.5× interquartile range from the hinges. Data points beyond this range are revealed as individual dots and represent outliers. (F) Publicly available NUP98::NSD1 chromatin immunoprecipitation-sequencing data reveal binding of NUP98::NSD1 to regions with proximal or distal enhancer-like signatures according to the ENCODE candidate cis-Regulatory Elements atlas ( GSE112928 ). CTL, control; Dox, doxycycline; FPKM, fragments per kilobase per million; H3K4me3, histone 3, lysine 4 trimethylation; KT, karyotype; mRNA, messenger RNA; RMA, robust multi-array analysis.
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Clec12a expression is regulated by NUP98::NSD1. (A) CLEC12A mRNA expression among different cytogenetic AML subgroups, derived from the Leucegene cohort (NUP98::NSD1, n = 7; CN-AML, n = 168; N/K-RASmut, n = 87; N/K-RASwt, n = 350; complex KT, n = 139; CBFB::MYH11, n = 29; CEBPAmut, n = 16; MLL:MLLT3, n = 42; PML::RARA, n = 15; RUNX1::RUNX1T1, n = 21; normal CD34 + , n = 17). Values are in log (FPKM). (B) CLEC12A mRNA expression among different AML KTs and mutations, derived from the Balgobind et al cohort ( GSE17855 ; NUP98::NSD1, n = 11; CN-AML, n = 39; MLL::MLT3, n = 47; N/K-RASmut, n = 41; N/K-RASwt, n = 196; CEBPAmut, n = 16; cKITmut, n = 18; FLT3-ITD, n = 48; CBFB::MYH11, n = 27; PML::RARA, n = 19; RUX1::RUNX1T1, n = 28; other, n = 45; unknown, n = 25; NPM1mut, n = 17). Values are in normalized microarray intensity (RMA). (C) Reverse <t>transcriptase</t> <t>PCR</t> revealing Clec12a mRNA expression in murine BM cells transduced with either NRASG12D, NUP98::NSD1, or NUP98::NSD1+NRASG12D (n = 3; mean ± standard error of the mean [SEM]). (D) Flow cytometric representation of Clec12a protein expression in murine hematopoietic BM cells transduced with NUP98::NSD1+NRASG12D. Cells were gated on GFP + BFP + . (E) Normalized Clec12a expression for NUP98 CTL and NUP98::NSD1 murine-expressing cells. Clec12a is highly expressed in NUP98::NSD1-expressing cells (off Dox) compared with NUP98 CTL cells and is strongly downregulated if NUP98-NSD1 expression is shut off (day 3_on_Dox and day 5_on_Dox). Data are presented as boxplots in which the center line represents the median, the boundaries of the box indicate the first (25th percentile) and third (75th percentile) quartiles, and the whiskers extend to 1.5× interquartile range from the hinges. Data points beyond this range are revealed as individual dots and represent outliers. (F) Publicly available NUP98::NSD1 chromatin immunoprecipitation-sequencing data reveal binding of NUP98::NSD1 to regions with proximal or distal enhancer-like signatures according to the ENCODE candidate cis-Regulatory Elements atlas ( GSE112928 ). CTL, control; Dox, doxycycline; FPKM, fragments per kilobase per million; H3K4me3, histone 3, lysine 4 trimethylation; KT, karyotype; mRNA, messenger RNA; RMA, robust multi-array analysis.
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Clec12a expression is regulated by NUP98::NSD1. (A) CLEC12A mRNA expression among different cytogenetic AML subgroups, derived from the Leucegene cohort (NUP98::NSD1, n = 7; CN-AML, n = 168; N/K-RASmut, n = 87; N/K-RASwt, n = 350; complex KT, n = 139; CBFB::MYH11, n = 29; CEBPAmut, n = 16; MLL:MLLT3, n = 42; PML::RARA, n = 15; RUNX1::RUNX1T1, n = 21; normal CD34 + , n = 17). Values are in log (FPKM). (B) CLEC12A mRNA expression among different AML KTs and mutations, derived from the Balgobind et al cohort ( GSE17855 ; NUP98::NSD1, n = 11; CN-AML, n = 39; MLL::MLT3, n = 47; N/K-RASmut, n = 41; N/K-RASwt, n = 196; CEBPAmut, n = 16; cKITmut, n = 18; FLT3-ITD, n = 48; CBFB::MYH11, n = 27; PML::RARA, n = 19; RUX1::RUNX1T1, n = 28; other, n = 45; unknown, n = 25; NPM1mut, n = 17). Values are in normalized microarray intensity (RMA). (C) Reverse transcriptase PCR revealing Clec12a mRNA expression in murine BM cells transduced with either NRASG12D, NUP98::NSD1, or NUP98::NSD1+NRASG12D (n = 3; mean ± standard error of the mean [SEM]). (D) Flow cytometric representation of Clec12a protein expression in murine hematopoietic BM cells transduced with NUP98::NSD1+NRASG12D. Cells were gated on GFP + BFP + . (E) Normalized Clec12a expression for NUP98 CTL and NUP98::NSD1 murine-expressing cells. Clec12a is highly expressed in NUP98::NSD1-expressing cells (off Dox) compared with NUP98 CTL cells and is strongly downregulated if NUP98-NSD1 expression is shut off (day 3_on_Dox and day 5_on_Dox). Data are presented as boxplots in which the center line represents the median, the boundaries of the box indicate the first (25th percentile) and third (75th percentile) quartiles, and the whiskers extend to 1.5× interquartile range from the hinges. Data points beyond this range are revealed as individual dots and represent outliers. (F) Publicly available NUP98::NSD1 chromatin immunoprecipitation-sequencing data reveal binding of NUP98::NSD1 to regions with proximal or distal enhancer-like signatures according to the ENCODE candidate cis-Regulatory Elements atlas ( GSE112928 ). CTL, control; Dox, doxycycline; FPKM, fragments per kilobase per million; H3K4me3, histone 3, lysine 4 trimethylation; KT, karyotype; mRNA, messenger RNA; RMA, robust multi-array analysis.

Journal: Blood Advances

Article Title: Clec12a is required for the pathogenesis of NUP98::NSD1 AML ∗

doi: 10.1182/bloodadvances.2024015739

Figure Lengend Snippet: Clec12a expression is regulated by NUP98::NSD1. (A) CLEC12A mRNA expression among different cytogenetic AML subgroups, derived from the Leucegene cohort (NUP98::NSD1, n = 7; CN-AML, n = 168; N/K-RASmut, n = 87; N/K-RASwt, n = 350; complex KT, n = 139; CBFB::MYH11, n = 29; CEBPAmut, n = 16; MLL:MLLT3, n = 42; PML::RARA, n = 15; RUNX1::RUNX1T1, n = 21; normal CD34 + , n = 17). Values are in log (FPKM). (B) CLEC12A mRNA expression among different AML KTs and mutations, derived from the Balgobind et al cohort ( GSE17855 ; NUP98::NSD1, n = 11; CN-AML, n = 39; MLL::MLT3, n = 47; N/K-RASmut, n = 41; N/K-RASwt, n = 196; CEBPAmut, n = 16; cKITmut, n = 18; FLT3-ITD, n = 48; CBFB::MYH11, n = 27; PML::RARA, n = 19; RUX1::RUNX1T1, n = 28; other, n = 45; unknown, n = 25; NPM1mut, n = 17). Values are in normalized microarray intensity (RMA). (C) Reverse transcriptase PCR revealing Clec12a mRNA expression in murine BM cells transduced with either NRASG12D, NUP98::NSD1, or NUP98::NSD1+NRASG12D (n = 3; mean ± standard error of the mean [SEM]). (D) Flow cytometric representation of Clec12a protein expression in murine hematopoietic BM cells transduced with NUP98::NSD1+NRASG12D. Cells were gated on GFP + BFP + . (E) Normalized Clec12a expression for NUP98 CTL and NUP98::NSD1 murine-expressing cells. Clec12a is highly expressed in NUP98::NSD1-expressing cells (off Dox) compared with NUP98 CTL cells and is strongly downregulated if NUP98-NSD1 expression is shut off (day 3_on_Dox and day 5_on_Dox). Data are presented as boxplots in which the center line represents the median, the boundaries of the box indicate the first (25th percentile) and third (75th percentile) quartiles, and the whiskers extend to 1.5× interquartile range from the hinges. Data points beyond this range are revealed as individual dots and represent outliers. (F) Publicly available NUP98::NSD1 chromatin immunoprecipitation-sequencing data reveal binding of NUP98::NSD1 to regions with proximal or distal enhancer-like signatures according to the ENCODE candidate cis-Regulatory Elements atlas ( GSE112928 ). CTL, control; Dox, doxycycline; FPKM, fragments per kilobase per million; H3K4me3, histone 3, lysine 4 trimethylation; KT, karyotype; mRNA, messenger RNA; RMA, robust multi-array analysis.

Article Snippet: Quantitative reverse transcriptase polymerase chain reaction (PCR) was performed using SYBR green (Qiagen) on a StepOne Plus cycler (Thermo Fisher Scientific) as previously described.

Techniques: Expressing, Derivative Assay, Microarray, Reverse Transcription, Transduction, ChIP-sequencing, Binding Assay, Control