Cytarabine (Ara-C) and Daunorubicin (Dnr) forms the backbone of acute myeloid leukemia (AML) therapy. Drug resistance and toxic side effects pose a major threat to treatment success and hence alternate less toxic therapies are warranted. NF-E2 related factor-2 (Nrf2), a master regulator of antioxidant response is implicated in chemoresistance in solid tumors. However, little is known about the role of Nrf2 in AML chemoresistance and the effect of pharmacological inhibitor brusatol in modulating this resistance. Primary AML samples with high ex-vivo IC50 to Ara-C, ATO, Dnr had significantly high NRF2 RNA expression. Gene-specific knockdown of NRF2 improved sensitivity to these drugs in resistant AML cell lines by decreasing the expression of downstream antioxidant targets of Nrf2 by compromising the cell’s ability to scavenge the ROS. Treatment with brusatol, a pharmacological inhibitor of Nrf2, improved sensitivity to Ara-C, ATO, and Dnr and reduced colony formation capacity. AML cell lines stably overexpressing NRF2 showed increased resistance to ATO, Dnr and Ara-C and increased expression of downstream targets. This study demonstrates that Nrf2 could be an ideal druggable target in AML, more so to the drugs that function through ROS, suggesting the possibility of using Nrf2 inhibitors in combination with chemotherapeutic agents to modulate drug resistance in AML.

Achieving early molecular response (EMR) has been shown to be associated with better event free survival in patients with chronic phase chronic myeloid leukemia (CP-CML) on Imatinib therapy. We prospectively evaluated the factors influencing the 2-year failure free survival (FFS) and EMR to imatinib therapy in these patients including day29 plasma Imatinib levels, genetic variants and the gene expression of target genes in imatinib transport and biotransformation. Patients with low and intermediate Sokal score had better 2-year FFS compared to those with high Sokal Score (p = 0.02). Patients carrying ABCB1-C1236T variants had high day29 plasma imatinib levels (P = 0.005), increased EMR at 3 months (P = 0.044) and a better 2 year FFS (P = 0.003) when compared to those with wild type genotype. This translates to patients with lower ABCB1 mRNA expression having a significantly higher intracellular imatinib levels (P = 0.029). Higher day29 plasma imatinib levels was found to be strongly associated with patients achieving EMR at 3 months (P = 0.022), MMR at 12 months (P = 0.041) which essentially resulted in better 2-year FFS (p = 0.05). Also, patients who achieved EMR at 3 months, 6 months and MMR at 12 months had better FFS when compared to those who did not. This study suggests the incorporation of these variables in to the imatinib dosing algorithm as predictive biomarkers of response to Imatinib therapy.

Although 70-80% of Acute Myeloid Leukemia (AML) patients achieve complete remission with the induction therapy consisting of a combination of Cytarabine (Ara-C) and Daunorubicin (Dnr), the overall survival is dismal with disease relapse, drug resistance and toxicities as the major causes of treatment failure. Single agent arsenic trioxide (ATO) used in acute promyelocytic leukemia (APL or AML-M3) is associated with relatively less toxicities which suggest the possibility of extending its use to elderly non M3-AML patients. But ATO trials in non-M3 AML were not encouraging enough to devise ATO as a better treatment option. NF-E2 related factor 2 (NRF2) has been recognised as one of the key molecules associated with resistance towards chemotherapeutic agents in cancer. Leukemic cells owing to their genomic instability and altered metabolism are known to maintain high reactive oxygen species (ROS) levels. NRF2 as a redox dependent transcription factor drives the expression of several antioxidant genes like NAD(P)H quinone oxidoreductase1 (NQO1), heme oxygenase1 (HMOX1), glutamate-cysteine ligase (GCL) and glutathione-S-transferases (GSTs) which coordinate to scavenge ROS. Although the role of NRF2 in resistance to cytarabine and daunorubicin has been reported previously, its role in ATO resistance in non M3-AML has not been addressed before. Also, the role of pharmacological inhibition of NRF2 in modulating resistance to ATO and other conventional therapeutic agents in AML has not been tested. To investigate whether NRF2 expression is associated with in-vitro sensitivity to chemotherapeutic drugs, NRF2 RNA expression in primary AML samples grouped according to their median Ara-C, Dnr and ATO IC50 (median IC50 6uM for Ara-C, 0.4µM for Dnr and 2.42µM for ATO) were compared. Primary AML samples with Ara-C, Dnr or ATO IC50 below median had significantly low NRF2 RNA expression compared to those above median (p=0.07, 0.004 and 0.01, respectively) (Fig1-A). Comparison of NRF2 expression in a subset of samples that were above median IC50 to both ATO and Dnr with those below median also showed similar trend. Flow cytometric evaluation of NRF2 expression in Ara-C, Dnr and ATO resistant AML cell lines (THP1 and U937) showed higher intracellular NRF2 levels (MFI=37.18 and 46.56) compared to sensitive cell lines (HL60 and MOLM13) (MFI=6.25 and 28.9). Treatment of resistant AML cell lines expressing high levels of NRF2 (THP1 and U937) with pharmacological inhibitor of NRF2 {Brusatol} followed by ATO (0.1-6uM), Ara-C (0.1uM-80uM) and Dnr (25nM-2000nM) improved their sensitivity to these drugs (Fig1-B) The effect of Brusatol in inhibiting NRF2 protein levels and its role in inducing apoptosis were also demonstrated by flow cytometry (Fig1-C). To further confirm the role of NRF2 in drug resistance, AML cell lines (MOLM13 and HL60) stably over expressing NRF2 was established. Overexpression was done using lentiviral expression system followed by blasticidin selection, further confirmed by immunoblot and quantitative real time PCR. The overexpressed cell lines showed increased resistance to ATO/ Dnr and Ara-C (Fig1-D) and up-regulation of NRF2 downstream targets compared to un-transduced cells (Fig1-E). Our results suggest that NRF2 plays a pivotal role in drug resistance and hence could be an ideal druggable target in AML, more so to the drugs that functions through ROS. Our data as well growing evidence from other malignancies suggest the possibility of using NRF2 inhibitors in combination with chemotherapeutic agents to combat drug resistance. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.

Early complications post hematopoietic stem cell transplantation (HSCT) such as sinusoidal obstruction syndrome (SOS) and graft versus host disease (GVHD) can be life threatening. Although several biomarkers have been identified to correlate with these complications and their response to treatment, these are yet to be used in clinical practice. Here, we evaluated circulating endothelial cells (CECs) (n = 26) and plasma biomarkers (ST2, REG3α, VCAM1, ICAM1, TIM3) (N = 210) at early time points, to determine their association with early complications post‐HSCT. Elevated CEC counts at the end of conditioning was associated with GVHD, indicating endothelial damage during HSCT. Plasma levels of REG3α, VCAM1, ICAM1, and TIM3 on day 14 (D14) and D14 ICAM1 and D28 ST2 were significantly higher in patients with SOS and aGVHD, respectively. Upon sub‐group analysis, D28 ST2, D14/D28 REG3α, and D14 ICAM1 levels were significantly higher in patients with gastrointestinal GVHD, while D28 ST2 was higher in those with skin/liver GVHD. High ST2 levels on D28 was significantly associated with non‐relapse mortality (NRM) and overall survival. Our results suggest that elevated ST2 levels on D28 could predict the likelihood of developing aGVHD and could influence NRM and OS.

The success of differentiation therapy is limited to acute promyelocytic leukemia (APL), and approaches to overcome the differentiation block in non-M3 AML have been unsuccessful. Nuclear hormone receptors (NHR) belong to ligand-inducible transcription factors that govern many cellular functions like differentiation, metabolism, and development. Retinoic Acid Receptor Alpha (RXRA) is a class of NHR that, when activated by all-trans retinoic acid (ATRA), successfully alleviates differentiation block in APL. To identify the NHRs/cofactors that could mediate or prevent differentiation in AML, we examined the differentially expressed NHRs and cofactors between ATRA sensitive (ATs) (NB4 and HL60) vs. ATRA resistant (ATr) AML cell lines (KG1a, Hel, K562, MV4-11, and OCI-AML3). Nuclear Receptor Interacting Protein 1 (NRIP1), a corepressor known to prevent transactivation of ligand-activated NHRs preferentially, was one of the top upregulated targets in the ATr cell lines (3.5 fold increase in RNA expression, figure 1a ). Immunoblot analysis also showed a significant increase in NRIP1 protein expression in the ATr than ATs cell lines (Figure 1b). Further, probing for NRIP1 expression in the publicly available TCGA and MILE AML study cohorts showed decreased NRIP1 expression in the APL cohort compared to other AML subtypes. Methylation profile from CCLE database of the NRIP1 promoter in AML cell lines showed ATs cell lines to be highly methylated compared to the ATr cell lines, suggesting the involvement of NRIP1 in mediating differentiation block in non-M3 AML (Figure 1c). To further dissect the role of NRIP1 in mediating this differentiation block, we carried out experiments in the AML cell line KG1a (having primitive blast features, high expression of NRIP1, and unresponsive to ATRA). Using CRISPR-cas9, we developed an NRIP1 knock-out (KO) cell line (Figure 1d). NRIP1 KO cell line showed a significant reduction in proliferation rate (Doubling time 26.2 vs. 36.5Hrs p<0.05). Further, cell cycle analysis revealed that NRIP1 KO leads to increased accumulation of cells in the G0 phase than in the S-phase (Figure 1e & f). We next assessed the sensitivity of the NRIP1 WT/KO cells to retinoic acids ATRA and bexarotene. Cells were treated with 1µM ATRA / bexarotene or in combination for 72 hours and evaluated for differentiation using CD11b marker by flow cytometry. NRIP1 KO alone leads to a marginal increase in basal CD11b expression compared to the WT cells (Mean CD11b expression 2.03% Vs 0.91%). ATRA treatment further increased the CD11b expression to 3.8% in KO cells compared to 1.6% in the WT cells. A similar increase in CD11b expression was observed in bexarotene-treated cells (3.7% Vs 1.24%). Combination of ATRA with bexarotene showed a 3-fold increase in CD11b expression in the KO cells compared to the WT (23.9% Vs 7.2%, Figure 1g). NRIP1 KO diminishes its repressive action on ligand-activated RARA (ATRA activated) and RXRA (Bexarotene-activated), thereby allowing synergistic differentiation induction by retinoic acids in AML cells. This study suggests a potential mechanism of differentiation inhibition mediated by corepressor NRIP1 in AML cells unresponsive to retinoic acids. Further in-depth analyses of molecular pathways governed by NRIP1 during ligand activation of NHRs are warranted to design differentiation therapies for AML. Figure 1 Figure 1. Disclosures Mathews: Christian Medical College: Patents & Royalties: US 2020/0345770 A1 - Pub.Date Nov.5, 2020; AML: Other: Co-Inventor.