Expression Profiling Of Nuclear Hormone Receptors In Myeloid Leukemia Reveals Potential Novel Drug Targets For Combination Therapy

Nuclear Hormone Receptors (NHRs) are a large superfamily of ligand dependent transcription factors regulating a plethora of genes involved in metabolism, growth and differentiation. Recent studies identified the overwhelming role played by NHR in determining hematopoiesis as well as HSC function and fate, suggesting that defective proliferation and maturation of progenitors in hematopoietic malignancies could be attributed to altered expression of these transcriptional regulators. NHRs are attractive but relatively unexplored drug targets and there is limited data on their expression profile in hematological malignancies. Our preliminary screening of AML cell lines and primary cells revealed differential expression of PPARg and RXRα among AML cell lines and decreased expression of these targets in resistant cell lines vs. sensitive ones (Fig.1a). We extended this study aiming to identify the expression pattern of all NHRs in myeloid malignancies, compare with ex-vivo chemoresistance and prognostic markers.

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Myeloid leukemia cell lines AML(n=12) carrying AML-ETO, Inv-16, PML-RAR fusion transcript, NPM1 or FLT3 mutation and CML(n=8), pooled primary samples harboring only FLT3 ITD mutation and only NPM1 mutation, CD34+ cells and total cellular RNA samples from normal donors were included in the study. Bone marrow samples from AML patients and peripheral blood samples from healthy volunteers were collected after informed consent. PBSC from normal donors (n=7) were enriched for CD34+ cells by magnetic enrichment. RNA extraction followed by purification and cDNA synthesis were done as per manufacturer’s recommendation. The expression of 42 NHRs and 42 co-regulators was profiled by qRT PCR using RT² PCR Profiler array (SABiosciences, Germany). Expression of each gene was normalized to the average of 5 housekeeping genes (GAPDH, b-actin, b2-microglobulin, HGPRT & RPLP0) and differential expression was calculated by normalizing this with normal total RNA samples to determine fold change. Data normalization and differential gene expression were computed using SABiosciences web-based analysis software. Ex-vivo cytotoxicity to cytarabine and daunorubicin was analyzed using MTT assay and IC50 was calculated using Adapt software. Based on Ara-C and Dnr IC50, AML samples were categorized as sensitive or resistant (IC50<6.25µM and >6.25µM for Ara-C;<0.5µM and >0.5µM for Dnr). FLT3-ITD and NPM1 mutations were screened using PCR followed by GeneScan methods.

Upon analysis we observed steroid receptors except PPARG, NR5A1, RXRG, NR1D2, glucocorticoid receptors, androgen receptors and their co-regulatory molecules were expressed across all cell lines. In contrast, expression of certain NHRs such as NR2E3, NR1I2, NR1H4, NR0B1, NR0B2, ESRRG, ESRRB, NR1D1 and the co-regulator HDAC7 were less or at undetectable levels, while estrogen receptors and its co-regulatory molecules except PPARGC1B, steroid receptors HNF4A, NR2F1, NR2F2, RARG and NR3C2 were moderately expressed across the cell lines (Fig.1b). Analysis also revealed significantly reduced expression of ESR2, ESRRG, NR2F2, NR2F6 and THRB and increased PPARG in AML compared to CML cell lines. Comparison between CD34+ progenitor population and well differentiated hematopoietic cells demonstrated >6 fold change in NR4A1 expression. Likewise, expression of several differentiation markers NR1H3, NR1D2, RORA, RORB, RARA and VDR were elevated in cells with increased degree of maturation. The receptor inducing terminal differentiation of erythrocytes THRA, had lesser expression (<60 Fold) in erythroleukemic cell lines K562, HEL and LAMA-84 compared to normal (Fig.1c). In Ara-C resistant cell lines, expression of NR1I3, AHR, and HNF4A were up-regulated while PPARG and RXRA were down-regulated. NPM1 positive cell lines and patient samples had increased expression of PPARG, PPARGC1A, NOTCH2, NR1H3 and NR2F2 than the FLT3 mutated group (Fig.1d) and similar expression of PPARG and RXRA was validated in NPM mutated primary AML blasts (Fig.1e).

Our comprehensive analysis of NHRs and its co regulatory molecules provides us with insights on NHR expression on differentiation and drug resistance across myeloid leukemia cell lines with various genetic and molecular characteristics. This could be further explored to identify potential novel drug targets to be used as combination therapy in myeloid leukemias to overcome drug resistance.