Page 266 - Haematologica March 2020

F. Chatonnet et al.
for a FOXM1-dependency of primary cancer cells as well as derived cell lines, FDI-6, a DNA binding inhibitor of FOXM1,42 was used. Primary MM cells were highly sensi- tive to FOXM1 inhibition compared to bone marrow microenvironment cells (Figure 4C). Overexpression of FAM72D in XG21 cells partially counteracted FDI-6- induced cell death (Online Supplementary Figure S8E), sug- gesting that FAM72D mediates part of FOXM1 effects on cell growth and survival.
In order to extend our findings to other cancer types, publicly available data were analyzed (http://www.cbiopor- tal.org/,43 https://xenabrowser.net/heatmap/) and indicated (i) that amplification of 1q21 is a highly prevalent event in breast cancer (Online Supplementary Figure S9A) and (ii) a higher expression of FAM72D within mutated TP53 patients (Online Supplementary Figure S9B). In agreement with FAM72D genes being targets of FOXM1, inhibition of FOXM1 in MCF-7 breast cancer cells has been shown to significantly reduce FAM72D, FAM72A and FAM72B expression.44 Hence, we next generated genome-wide maps of 5hmC in MCF-7 cells and observed that, as in MM cells, the upstream region of FAM72D was highly hydroxymethylated (Online Supplementary Figure S9C),
AB
suggesting a similar regulation of this gene between MM and breast cancer cells. To investigate a putative function of FAM72D in mitosis, FAM72D was knocked-down by transfection of siRNAs in MCF-7 cells and mitotic anom- alies were analyzed. Data revealed that a reduction in FAM72D levels led to a higher occurrence of defects such as misaligned chromosomes, lagging chromatids and micronuclei (Online Supplementary Figure S9D), suggesting that FAM72D helps maintain mitotic fidelity.
High FAM72 expression is associated with resistance to bortezomib and sensitivity to histone deacetylase/decitabine inhibitors (HDACi/DNMTi)
Since FAM72D expression is associated with a poor out- come in MM, we investigated whether high FAM72 expression could be related with drug resistance in MM. Correlating FAM72 gene expression with response to con- ventional drugs (bortezomib, melphalan, lenalidomide, dexamethasone and panobinostat) in HMCL, we identi- fied that high FAM72 expression levels are associated with resistance to bortezomib (n=12, P=0.049) (Figure 5A). These observations are consistent with the fact that high FAM72 expression is associated with a poor prognosis in a
C
P=0.0487
P=0.035
Figure 5. FAM72 expression levels predict multiple myeloma cell sensitivity to drugs. (A) 12 HMCL were cultured with graded concentrations of Boterzomib for four days and IC50 were calculated with mean values of five experiments determined on sextuplet culture wells. High FAM72 expression (Affymetrix microarrays) was sig- nificantly correlated with resistance to bortezomib. (B) Histone deacetylase/decitabine inhibitors (HDACi/DNMTi) induce a significant downregulation of FAM72 and FOXM1 in multiple myeloma (MM). Nine HMCL were treated for seven days with decitabine (DNMTi) and TSA during the last 24 hours and gene expression was assessed using Affymetrix U133P microarrays. (C) FAM72 expression predicts 5azacitidine/SAHA combination sensitivity of primary myeloma cells of patients. Mononuclear cells from tumor samples of 17 patients with MM were cultured for 4 days in the presence of IL-6 (2 ng/mL) with or without 2 mM 5azacitidine and 300 nM SAHA. At day 4 of culture, the count of viable MMC was determined using CD138 staining by flow cytometry. Samples from Figure 6C and Online Supplementary Figure S10C were grouped as FAM72high or low and FOXMhigh or low and correlation coefficients between FAM72 or FOXM1 expression and the per- centage of living MM cells were calculated with GraphPad Prism.
Gene Correlation P-value
780
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