Identification of SHMT2 and ADAM28 as potential mediators of NFIC pro-growth effects in AML Free

Acute myeloid leukemia (AML) is a heterogeneous group of hematological malignancies characterized by the accumulation of clonal, abnormally differentiated blasts in the bone marrow. For most patients, prognosis remains poor and a large proportion relapse. Using a small cohort of AML blasts, we previously showed using proteomic analysis that the expression of the transcription factor, nuclear factor 1C (NFIC) was elevated in 69% (9/13) of AML patients compared to normal hematopoietic stem and progenitor cells (HSPC). Further, that ectopic expression of NFIC in HSPC promoted growth and monocytic development and that NFIC knockdown (KD) reduced the growth of AML blasts. Here we show the clinical significance of NFIC expression, and the molecular mechanisms and cellular pathways utilized by NFIC to promote AML growth.

We first determined NFIC protein expression in a larger, more heterogeneous group of AML blasts obtained from the UK-MRC AML15 clinical trial. We analyzed the protein composition of 91 primary AML blasts and compared NFIC expression to normal human CD34⁺ HSPC, CD14⁺ monocyte and CD15⁺ granulocyte controls using mass spectrometry. Consistent with our previous data, NFIC protein expression was detected in 68% (62/91) of AML blasts and undetectable in normal blood cell populations, a result confirmed by western blotting.

To determine the clinical significance of NFIC expression, we initially analyzed AML patient blast mRNA expression data using the BEAT AML dataset. Comparing European Leukemia Network prognosis groups, NFIC mRNA expression was significantly higher among AML patients with favorable prognosis compared to intermediate and adverse groups. NFIC expression was strongest among core-binding factor AML (CBFB::MYH11 and RUNX1::RUNX1T1) and acute promyelocytic leukemia. The presence of NPM1 or FLT3-ITD mutations had no clinically significant effect on NFIC expression. NFIC expression did not correlate with patient age, gender, white blood cell count or French-American-British grouping suggesting elevated NFIC expression is an independent prognostic factor.

To identify the mechanisms utilized by NFIC to support AML cell growth, we analyzed NFIC's effects on gene expression and chromatin organization. The effect of NFIC KD was analyzed in THP-1 cells using mRNA sequencing coupled with promotor capture Hi-C (PCHi-C) sequencing and compared to scrambled control (SCR). Enrichment analysis of differentially expressed genes (log2FC≥1) revealed that NFIC KD in AML cells promoted expression of genes involved in nucleosome assembly and chromatin silencing. Meanwhile, NFIC KD suppressed expression of genes involved in extracellular matrix (ECM) organization, PI3K-Akt pathway signaling and amino acid synthesis, transport and metabolism. PCHi-C sequencing identified 192 differential chromatin interactions involving 256 unique gene promotors. To identify the upstream functional targets of NFIC, we interrogated our list of ~1800 differentially expressed genes, obtained by mRNA sequencing, for genes with differential promotor interactions. This yielded 16 potential functional targets of NFIC in AML cells, including ADAM28, which has previously been shown to regulate the growth, migration and invasion of AML cells. To determine which of these targets showed correlative expression with NFIC in AML patients, we carried out a Pearson correlation analysis of mRNA data from the TARGET AML dataset. Of the 16 genes, 4 (PCOLCE2, SAMD10, SHMT2 and ZNF512B) correlated with NFIC expression in AML patients (R² > 0.3). Finally, we validated differential expression of SHMT2 and ADAM28 (included due to its previous association with AML) in NFIC KD THP-1 cells compared to SCR using western blotting. No difference in PCOLCE2 expression was detected and expression of SAMD10 and ZNF512B were undetectable in all samples using western blotting.

In summary we show that NFIC overexpression in AML cells primarily induces PI3K-Akt pathway signaling, amino acid metabolism and ECM reorganization to support AML growth providing new potential avenues for therapeutic targeting of NFIC signaling in AML. Further, we identify SHMT2 and ADAM28 as potential upstream targets of NFIC that may be key in mediating NFIC's pro-growth effects in AML and are currently determining whether ectopic expression of these proteins can rescue the effect of NFIC knock down.