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Despite the efficacy of tyrosine kinase inhibitors (TKIs) in chronic myeloid leukemia (CML), malignant long-term hematopoietic stem cells (LT-HSC) persist as a source of relapse. However, LT-HSC are heterogenous and the most primitive, drug-resistant LT-HSC subpopulations are not well characterized. In normal hematopoiesis, self-renewal and long-term reconstitution capacity is enriched within LT-HSCs with low c-Kit expression (c-KitLow). Here, using a transgenic CML mouse model, we found that long-term engraftment and leukemogenic capacity were restricted to c-KitLow CML LT-HSC. CML LT-HSC demonstrated enhanced differentiation with expansion of mature progeny following exposure to the c-Kit ligand, stem cell factor (SCF). Conversely, SCF deletion led to depletion of normal LT-HSC but increase in c-KitLow and total CML LT-HSC with reduced generation of mature myeloid cells. CML c-KitLow LT-HSC showed reduced cell cycling, and expressed enhanced quiescence and inflammatory gene signatures. SCF administration led to enhanced depletion of CML primitive progenitors but not LT-HSC after TKI treatment. Human CML LT-HSC with low or absent c-Kit expression were markedly enriched after TKI treatment. We conclude that CML LT-HSC expressing low c-Kit levels are enriched for primitive, quiescent, drug-resistant leukemia initiating cells and represent a critical target for eliminating disease persistence.

Background Acute cutaneous graft-versus-host disease (acGVHD) following haematopoietic stem cell transplant (HSCT) is common but difficult to distinguish from other causes of rash. Plasma elafin has been proposed as a diagnostic and prognostic biomarker of skin GVHD. Aim To evaluate the role of plasma elafin as a biomarker in acGVHD in an Indian population. Methods Plasma elafin was evaluated in a prospective study of HSCT recipients, conducted over 2 years, taking measurements at baseline and at onset of skin rash after HSCT. Patients were categorized into those with GVHD rash, those with non-GVHD rash and those with no rash and the three groups were compared. Results Two hundred and sixty-one patients with a median age of 16 years (range 1–61 years) and a male predominance (175 : 86 M/F) underwent HSCT during the study period: 56 patients in the GVHD group, 49 in the non-GVHD group and 156 in the no-rash group. The median baseline elafin was similar in all three groups. At the onset of rash, median elafin level was similar between GVHD and non-GVHD rash (34 549 vs. 32 077 pg/mL; P = 0.58) and between GVHD and no rash (34 549 vs. 26 197 pg/mL; P = 0.08). A rise in elafin from baseline was significantly different between GVHD and no rash (P < 0.001) but not between GVHD and non-GVHD rash (P = 0.44). Conclusion The utility of plasma elafin as a biomarker of skin GVHD is very limited. Plasma elafin, although elevated in cutaneous GVHD, is not helpful in distinguishing between GVHD rash and other causes of rash following HSCT.

Chronic myeloid leukemia (CML) results from hematopoietic stem cell transformation by the BCR-ABL kinase. Despite the success of BCR-ABL tyrosine kinase inhibitors (TKIs) in treating CML patients, leukemia stem cells (LSCs) resist elimination and persist as a major barrier to cure. Previous studies suggest that overexpression of the sirtuin 1 (SIRT1) deacetylase may contribute to LSC maintenance in CML. Here, by genetically deleting SIRT1 in transgenic CML mice, we definitively demonstrated an important role for SIRT1 in leukemia development. We identified a previously unrecognized role for SIRT1 in mediating increased mitochondrial oxidative phosphorylation in CML LSCs. We showed that mitochondrial alterations were kinase independent and that TKI treatment enhanced inhibition of CML hematopoiesis in SIRT1-deleted mice. We further showed that the SIRT1 substrate PGC-1α contributed to increased oxidative phosphorylation and TKI resistance in CML LSCs. These results reveal an important role for SIRT1 and downstream signaling mechanisms in altered mitochondrial respiration in CML LSCs.

Simple Summary The Hedgehog signaling pathway is aberrantly activated in many myeloid malignancies, and pathway inhibition is clinically beneficial in specific patients with acute myeloid leukemia. However, even with the approval of these agents, the role of Hedgehog signaling in other myeloid disorders is less clear. In this review, we summarize the laboratory studies that have examined Hedgehog signaling in normal and malignant hematopoiesis as well as the clinical studies that have been carried out in several myeloid leukemias. Finally, we explore potential strategies to further expand the use of pathway inhibitors as therapies for these diseases. Abstract Myeloid malignancies arise from normal hematopoiesis and include several individual disorders with a wide range of clinical manifestations, treatment options, and clinical outcomes. The Hedgehog (HH) signaling pathway is aberrantly activated in many of these diseases, and glasdegib, a Smoothened (SMO) antagonist and HH pathway inhibitor, has recently been approved for the treatment of acute myeloid leukemia (AML). The efficacy of SMO inhibitors in AML suggests that they may be broadly active, but clinical studies in other myeloid malignancies have been largely inconclusive. We will discuss the biological role of the HH pathway in normal hematopoiesis and myeloid malignancies and review clinical studies targeting HH signaling in these diseases. In addition, we will examine SMO-independent pathway activation and highlight potential strategies that may expand the clinical utility of HH pathway antagonists.

DNA methylation regulates gene transcription and is involved in various physiological processes in mammals, including development and hematopoiesis. It is catalyzed by DNA methyltransferases including Dnmt1, Dnmt3a, and Dnmt3b. For Dnmt3b, its effects on transcription can result from its own DNA methylase activity, the recruitment of other Dnmts to mediate methylation, or transcription repression in a methylation-independent manner. Low-frequency mutations in human DNMT3B are found in hematologic malignancies including cutaneous T-cell lymphomas, hairy cell leukemia, and diffuse large B-cell lymphomas. Moreover, Dnmt3b is a tumor suppressor in oncogene-driven lymphoid and myeloid malignancies in mice. However, it is poorly understood how the different Dnmt3b activities contribute to these outcomes. We modulated Dnmt3b activity in vivo by generating Dnmt3b+/− mice expressing one wild-type allele as well as Dnmt3b+/CI and Dnmt3bCI/CI mice where one or both alleles express catalytically inactive Dnmt3bCI. We show that 43% of Dnmt3b+/− mice developed T-cell lymphomas, chronic lymphocytic leukemia, and myeloproliferation over 18 months, thus resembling phenotypes previously observed in Dnmt3a+/− mice, possibly through regulation of shared target genes. Interestingly, Dnmt3b+/CI and Dnmt3bCI/CI mice survived postnatal development and were affected by B-cell rather than T-cell malignancies with decreased penetrance. Genome-wide hypomethylation, increased expression of oncogenes such as Jdp2, STAT1, and Trip13, and p53 downregulation were major events contributing to Dnmt3b+/− lymphoma development. We conclude that Dnmt3b catalytic activity is critical to prevent B-cell transformation in vivo, whereas accessory and methylation-independent repressive functions are important to prevent T-cell transformation.

Background The transfusion of platelets is an important therapeutic strategy in bleeding patients with thrombocytopenia. However, some chronically transfused patients fail to achieve the appropriate platelet count increment following transfusion due to the presence of platelet alloantibodies. Objectives The aims of this research were to study the prevalence of platelet alloimmunisation and to characterise the platelet-reactive (PR) antibodies in haematology patients refractory to platelet transfusions in an Indian setting. Patients and methods A total of 80 patients with a prior history of multiple transfusions (minimum of five cellular transfusions) were included in the study if they did not achieve an adequate corrected count increment within 24 h of the platelet transfusion. Patients with non-immunological causes of platelet refractoriness were excluded from the study. The test was performed on a blood sample of 4 mL of Ethylenediaminetetraacetic acid (EDTA) blood sample in which plasma was separated and stored at -80 °C and underwent batch testing in PAK-2LE. Results The overall prevalence of platelet alloimmunisation in our study was 60%. Of the 48 patients who were detected to have platelet antibodies, the combination of anti-human leucocyte antigen (HLA) and platelet-specific (PS) antibodies together constituted the majority of 54·2%. The overall prevalence of anti-HLA antibodies was 51·25% and of PS antibodies was 41·25% in the total study population of 80. Conclusion The overall prevalence of PS antibodies in our study was greater than that reported by other groups in India and other countries. This needs to be considered, particularly in the management of patients refractory to platelet transfusions, where HLA-matched platelets constitute current best practice.

SummaryIntroduction The hallmark of chronic myeloid leukemia (CML) is the presence of Philadelphia chromosome, its resultant fusion transcript (BCR-ABL1), and fusion protein (p210). Alternate breakpoints in BCR (m-bcr, μ-bcr, and others) or ABL1 result in the expression of few rare fusion transcripts (e19a2, e1a2, e13a3, e14a3) and fusion proteins (p190, p200, p225) whose exact clinical significance remains to be determined. Methods Our study was designed to determine the type and frequency of BCR-ABL1 fusion transcripts in 1260 CML patients and to analyze the prognosis and treatment response in patients harboring rare BCR-ABL1 fusion transcripts. Results The frequency of various BCR-ABL1 fusion transcripts was as follows: e14a2 (60%), e13a2 (34.3%), e1a2 (1.2%), e1a2 + e13a2 (2.0%), e1a2 + e14a2 (1.8%), e19a2 (0.3%), and e14a3 (0.3%). CML patients with e1a2 transcripts had higher rates of disease progression, resistance, or suboptimal response to imatinib and failed to achieve major molecular response. Conclusion Characterization of the specific fusion transcript in CML patients is important owing to the difference in prognosis and response to therapy in addition to the conventional need for monitoring treatment response. CML patients with e1a2 transcripts have to be closely monitored due to the high incidence of disease progression and treatment resistance/failure.

Introduction Type 3 von Willebrand disease (VWD) is the rare and most severe form of VWD which results from a near-complete deficiency of the von Willebrand factor (VWF). This study evaluates in detail the molecular pathology of type-3 VWD in India. One hundred and two patients from 90 families were evaluated. Patients and methods Phenotypic data, including bleeding scores (BS), were documented using structured questionnaires. Diagnosis of type 3 VWD was based on undetectable VWF antigen levels in the plasma. Genomic DNA from these patients was screened for mutations in VWF gene. Structural modeling and expression studies were carried out for missense mutations. Results Out of 102 patients, mutations could be identified in 91% (n = 93). Fifty-five different gene variants were identified. Thirty-four (61.8%) were novel. Mutations could be identified in both the alleles in 90 patients, while no causative mutation could be identified in 9 patients; twenty-four (23.5%) patients had mutations clustered in the propeptide region of VWF. Interestingly, five mutations accounted for the defects in 37/93 (39.8%) patients. Structural analysis and in vitro studies on missense mutations imply impaired processes associated with secretion of VWF. Conclusion This study is one of the largest series to define the molecular basis of type-3 VWD.

Background: Variation in terms of outcome and toxic side effects of treatment exists among acute myeloid leukemia (AML) patients on chemotherapy with cytarabine (Ara-C) and daunorubicin (Dnr). Candidate Ara-C metabolizing gene expression in primary AML cells is proposed to account for this variation. Methods: Ex vivo Ara-C sensitivity was determined in primary AML samples using MTT assay. mRNA expression of candidate Ara-C metabolizing genes were evaluated by RQPCR analysis. Global gene expression profiling was carried out for identifying differentially expressed genes between exvivo Ara-C sensitive and resistant samples. Results: Wide interindividual variations in ex vivo Ara-C cytotoxicity were observed among samples from patients with AML and were stratified into sensitive, intermediately sensitive and resistant, based on IC50 values obtained by MTT assay. RNA expression of deoxycytidine kinase (DCK), human equilibrative nucleoside transporter-1 (ENT1) and ribonucleotide reductase M1 (RRM1) were significantly higher and cytidine deaminase (CDA) was significantly lower in ex vivo Ara-C sensitive samples. Higher DCK and RRM1 expression in AML patient's blast correlated with better DFS. Ara-C resistance index (RI), a mathematically derived quotient was proposed based on candidate gene expression pattern. Ara-C ex vivo sensitive samples were found to have significantly lower RI compared with resistant as well as samples from patients presenting with relapse. Patients with low RI supposedly highly sensitive to Ara-C were found to have higher incidence of induction death (p = 0.002; RR: 4.35 [95% CI: 1.69–11.22]). Global gene expression profiling undertaken to find out additional contributors of Ara-C resistance identified many apoptosis as well as metabolic pathway genes to be differentially expressed between Ara-C resistant and sensitive samples. Conclusion: This study highlights the importance of evaluating expression of candidate Ara-C metabolizing genes in predicting ex vivo drug response as well as treatment outcome. RI could be a predictor of ex vivo Ara-C response irrespective of cytogenetic and molecular risk groups and a potential biomarker for AML treatment outcome and toxicity. Original submitted 22 December 2014; Revision submitted 9 April 2015

Mixed chimerism (MC) occurs frequently after allogeneic hematopoietic stem cell transplantation (HSCT) for thalassemia major (TM) and may be associated with rejection. We report the outcome of MC in 132 TM patients conditioned with Busulphan/Cyclophosphamide, who had successful engraftment and had ⩾1 year follow-up. Chimerism was first assessed at day +28, then every 3-9 months or more frequently if there was MC. If rejection was suspected, immunosuppression was stopped and donor-lymphocyte infusion (DLI) was given if there was no response. Among 132 patients, aged 7 years (range: 2-24), 46/132 (34.8%) had MC in the first year, 32/46 (69.6%) at day +28 and another 14 (30%) between day +28 and 1 year post HSCT. MC was quantified at level 1 (residual host chimerism (RHC)10%) in 20 (43.5%), level II (RHC 10-25%) in 14 (30.4%) and level III (RHC25%) in 12 (26.1%). On tapering immunosuppression, 15 (32.6%) developed acute GvHD and 8 (17.4%) had chronic GvHD with reversal to complete chimerism (CC). DLI was administered to 5/46 (10.9%), 1 evolved to CC but 4 rejected the graft. At median follow-up of 60 months (range: 16-172), 20/46 (43.5%) had CC, 18/46 (39.1%) had persistent MC with hemoglobin of 11.5 g/dL (range: 8.4-13.6), whereas 8 (17.4%) rejected the graft. Close monitoring and early intervention is needed with increasing recipient chimerism. Novel strategies are required for preventing graft rejection.

DNA methylation regulates gene transcription and it is involved in various physiological processes in mammals including development and hematopoiesis. It is catalyzed by several DNA methyltransferases, including DNMT1, DNMT3A and DNMT3B that not only mediate methylation but also interact with a number of repressive proteins, thereby contributing to transcriptional repression in methylation-independent manner. Dnmt3b is critical gene in mouse embryogenesis whose loss results in embryonic lethality around E13.5. It also functions as a tumor suppressor in lymphoid and myeloid malignancies in mice. The extent to which Dnmt3b's catalytic activity is involved in these processes is unclear. To address the role of catalytic activity in embryogenesis and tumorigenesis, we generated mice carrying a catalytically inactive allele of Dnmt3b (Dnmt3bCI) by introducing a double amino acid substitution in enzyme active center (P656V; C657D) in endogenous Dnmt3b locus. By interbreeding Dnmt3b+/CI mice we found that homozygous mutant Dnmt3bCI/CImice survived embryonic development and had a long life span. Such results demonstrated that catalytic activity of Dnmt3b is, surprisingly, dispensable for both pre- and post-natal development. The presence of catalytically inactive Dnmt3b rescued most methylation changes observed in the absence of Dnmt3b in embryogenesis suggesting that Dnmt3b plays a role of accessory protein for methylation mediated by other Dnmts. In contrast, the absence of Dnmt3b's catalytic activity promoted MYC/Ras-induced transformation of mouse embryonic fibroblasts in vitro, and accelerated both MYC-induced T-cell lymphomagenesis and MLL-AF9-induced acute myeloid leukemia. Global gene expression and methylation profiling revealed a number of deregulated events specific for MYC;Dnmt3bCI/CI, resulting in aberrant activation of IL7-R and STAT3 and downregulation of putative tumor suppressor genes including Cdkn2c and Dusp6. Altogether, our data suggest that Dnmt3b's methyltransferase activity is dispensable for mouse development but critical to prevent hematologic malignancies by controlling events involved in cellular transformation. Disclosures No relevant conflicts of interest to declare.

The FLT3-ITD mutation occurs in 25-30% of AML patients, and is associated with increased relapse rate and poor survival. FLT3 tyrosine kinase inhibitors (TKI) have shown encouraging results, but responses are not sustained. Therefore, there is considerable interest in understanding mechanisms of resistance of FLT3-ITD AML stem cells after TKI treatment. Autophagy is an important cellular response that maintains cell survival under stress. Here we investigated the regulation of autophagy in resistance of FLT3-ITD+ AML stem cells to FLT3 TKI treatment. MV4-11 and Molm13 FLT3-ITD AML cells were transduced with GFP-LC3-RFP-LC3 ΔG constructs to measure autophagic flux. Autophagyhigh AML cells showed slower growth(p We tested the effect of Lys05 in vivo by treating NSG mice engrafted with primary FLT3-ITD AML cells with vehicle, AC220 (10mg/kg), Lys05 (20mg/kg) or combination for 3 weeks. Lys05 treatment reduced human CD45+ cell engraftment (8.7% vs 17%, p=0.03), and reduced CD45+CD33+ myeloid cells in BM and spleen (BM: 42% vs 81%, p We conclude that autophagy inhibition enhances sensitivity of FLT3-ITD+ AML stem cells to TKI treatment both in vitro and in vivo. We demonstrate important roles for autophagy in regulating p53 levels and cell cycle in FL3-ITD AML cells. These studies support further development of strategies to target autophagy and related pathways to enhance efficacy of TKI in eliminating AML stem cells. Disclosures No relevant conflicts of interest to declare.

Cytosine methylation is involved in a variety of biological processes, including development, X-chromosome inactivation, imprinting, and suppressing unwanted transcription. DNA methylation in mammalian cells is catalyzed by several DNA methyltransferases, including DNMT1, DNMT3A and DNMT3B. In addition to DNA methylation catalysis, these enzymes interact with a number of repressive proteins including HDACs, thereby repressing transcription in methylation-independent manner. The extent to which methylation-dependent and independent activities play roles in biological processes remains poorly understood. Here we examined the role of methyltransferase (MT) activity of Dnmt3b in normal mouse embryogenesis and in prevention of hematologic malignancies. Loss of Dnmt3b in mice is embryonically lethal between 10.5-12.5 dpc. Dnmt3b was also identified as tumor suppressor in murine models of lymphoid and myeloid malignancies. To address whether Dnmt3b’s catalytic activity is important for mouse embryogenesis and prevention of hematologic malignancies, we generated mice bearing a conventional Dnmt3bCI allele using CRISPR/Cas9 system. This catalytically inactive allele of Dnmt3b (Dnmt3bCI) was generated by a double amino acid substitution in enzyme active center (P656V and C657D) abolishing its ability to perform transfer of methyl group onto cytosine. Surprisingly, Dnmt3bCI/CI mice were born at similar ratios as wild-type littermates and lived over 12 months suggesting that Dnmt3b is dispensable for both pre- and post-natal development. Importantly, MYC-induced T-cell lymphomagenesis was accelerated in the absence of Dnmt3b’s methyltransferase activity in MYC;Dnmt3bCI/CI mice. Global gene expression and methylation profiling revealed a number of deregulated events specific for MYC;Dnmt3bCI/CI, resulting in activation of Pak, Fgfr and Igf2 signaling and downregulation of tumor suppressors (e.g. Lrp12, Dusp6 and Marcks). The lack of Dnmt3b catalytic activity also accelerated a development of acute myeloid leukemia induced by MLL-AF9 overexpression in mice. Altogether, our data suggest that Dnmt3b’s MT activity is dispensable for mouse development but critical to prevent hematologic malignancies. Citation Format: Katarina Lopusna, Pawel Nowialis, Staci L. Haney, Ajay Abraham, Jana Opavska, Rene Opavsky. Critical role of Dnmt3b catalytic activity in prevention of oncogene-induced leukemias and lymphomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4339.

Nucleophosmin (NPM1) gene mutations are among the most studied genetic lesions in acute myeloid leukemia (AML) and are associated with a good prognosis. The frequency of exon 12 NPM1 mutation in ad...

Congenital fibrinogen deficiency is an extremely rare (1:1 000 000) hereditary bleeding disorder caused by defects in genes coding for fibrinogen Aα-, Bβ- and γ-chains, respectively. We report here the molecular basis of fibrinogen deficiency in a large series of patients from India. Twenty-seven patients with clinical features suggestive of fibrinogen deficiency and with prolonged plasma clotting times and low fibrinogen levels were studied. Genomic DNA was screened for mutations in the fibrinogen alpha (FGA), beta (FGB), gamma (FGG) genes by PCR and conformation sensitive gel electrophoresis. Fourteen different disease-causing mutations including frameshifts (51.9%), splice site (22.2%), missense (18.5%) and nonsense mutation (7.4%) were identified in 27 patients. Thirteen of them were novel, including seven frameshifts (fibrinogen Aα: p.Asp296 fs*59, p.Thr466 fs*17 and p.Lys575 fs*74; fibrinogen Bβ: p.Gly414 fs*2 and fibrinogen γ: p.Ser81 fs*5, p.Lys185 fs*13 and p.Asp278_279 fs*17), three splice site mutations (FGA gene c.364+1G>A; c.510+2 T>G; FGB gene c.851+1G>A), two missense substitutions (fibrinogen Bβ: p.Gly288Ser; p.Arg445Thr) and a nonsense mutation in fibrinogen Aα (p.Tyr127*). Two common mutations (FGA: c.364+1G>A, n = 6, FGG: p.Lys185 fs*13, n = 7) affecting 13 patients were identified in this series, suggesting that these mutations could be screened first in Indian patients with fibrinogen deficiency. The molecular data presented here is the largest series of patients with fibrinogen deficiency reported so far, adding significantly to the mutation database of this condition. It also helps create an algorithm for its genetic diagnosis in India.

Introduction: Drug resistance and relapse are considered to be the major reasons for treatment failure in acute myeloid leukemia (AML). There is limited data on the role of ABC transporter expression on in vitro sensitivity to cytarabine (Ara-C) and daunorubicin (Dnr) in primary AML cells. Patients & methods: RNA expression levels of 12 ABC transporters were analyzed by real-time quantitative PCR in 233 de novo adult acute myeloid leukemia patients. Based on cytarabine or Dnr IC50, the samples were categorized as sensitive, intermediate and resistant. Role of candidate ABC transporter RNA expression on in vitro cytotoxicity, treatment outcome post therapy as well as the influence of various prognostic markers on ABC transporter expression were analyzed. Results: Expression of ABCC3 and ABCB6 were significantly higher in Dnr-resistant samples when compared with Dnr-sensitive samples. Increased ABCC1 expression was associated with poor disease-free survival in this cohort of patients. Conclusion: This comprehensive analysis suggests ABCC1, ABCC3, ABCB6 and ABCA5 as probable targets which can be modulated for improving chemotherapeutic responses.

Summary. Data on the clinical manifestations of patients with clotting factor defects other than Haemophilia A, B and von Willebrand disease are limited because of their rarity. Due to their autosomal recessive nature of inheritance, these diseases are more common in areas where there is higher prevalence of consanguinity. There is no previous large series reported from southern India where consanguinity is common. Our aim was to analyze clinical manifestations of patients with rare bleeding disorders and correlate their bleeding symptoms with corresponding factor level. Data were collected in a standardized format from our centre over three decades on 281 patients who were diagnosed with rare bleeding disorders (fibrinogen, prothrombin, factor V (FV), FVII, FX, FXI, FXIII and combined FV or FVIII deficiency). Patients with liver dysfunction or those on medications which can affect factor level were excluded. All patients with 1% factor coagulant activities among all disorders, except for few symptoms in FVII and FX deficiency. An international collaborative study is essential to find out the best way of classifying severity in patients with rare bleeding disorders.

The FLT3-ITD mutation occurs in 25-30% of AML patients, and is associated with increased relapse rate and poor survival. FLT3 tyrosine kinase inhibitors (TKI) have shown encouraging results, but responses are not sustained in most patients. Therefore there is considerable interest in understanding mechanism contributing to resistance and preservation of FLT3-ITD AML stem cells after TKI treatment. Autophagy is an important cellular response that maintains cell survival under stress. Here we investigated the regulation of autophagy in FLT3-ITD+ AML cells, and its role in resistance to FLT3 TKI treatment. Expression of a FLT3-ITD construct in FLT3-ITD negative OCI-AML3 cells using lentiviral vectors resulted in increased autophagic flux compared with wild-type FLT3 or non-transduced controls. However, treatment of FLT3-ITD+ MV4-11 and Molm13 AML cells, or primary FLT3-ITD+ CD34+ cells, with the potent FLT3 TKI AC220 (Quizartinib) did not significantly alter autophagic flux. These results suggest that FLT3-ITD expression increases autophagy but in a kinase-independent manner. MV4-11 and Molm13 cells were transduced with the GFP-LC3-RFP-LC3 ΔG construct (Kaizuka T, et al,2016) to measure autophagic flux. Autophagyhigh AML cells showed lower growth rate compared with Autophagylow cells (0.65, p=0.025), and reduced sensitivity to inhibition by AC220 (0.49 vs 0.71, p=0.003). Treatment of MV4-11 and Molm13 cells with the potent autophagy inhibitor Lys05 in combination with AC220 resulted in significantly enhanced inhibition of AML cell growth (MV4-11: 0.83 vs 0.54, p Induction of autophagy by starvation or mTOR inhibitor treatment in MOLM13 and MV4-11 cells was associated with reduced p53 expression, whereas autophagy inhibition by chloroquine or SAR405, a PIK3C3/Vps34 inhibitor, was associated with increased p53 expression. Starvation-mediated autophagy in FLT3-ITD+ AML cells was significantly increased upon p53 knockdown, suggesting that p53 has an inhibitory effect on autophagy. Enhanced inhibition of FLT3-ITD+ AML cell growth by autophagy inhibition in combination with AC220 treatment was prevented by shRNA-mediated knockdown of p53, suggesting that effects of autophagy inhibition on FLT3-ITD+ AML cell growth were p53 dependent. We conclude that FLT3-ITD expression enhances autophagic flux in FLT3-ITD+ AML cells in a kinase-independent manner, and that increased autophagy contributes to TKI resistance of FLT3-ITD+ AML cells. We show that autophagy inhibition can enhance sensitivity of FLT3-ITD+ AML stem cells to TKI treatment. Finally, we demonstrate important roles for autophagy in regulating p53 levels, and conversely for p53 in regulating autophagy and the effects of autophagy inhibition in FLT3-ITD+ AML cells. These studies support further development of strategies for targeting of autophagy and related pathways to enhance efficacy of TKI treatment in eliminating AML stem cells. Disclosures No relevant conflicts of interest to declare.

Despite the success of BCR-ABL tyrosine kinase inhibitors (TKIs) in inducing remission and prolonging survival of CML patients, cures remain elusive because of persistence of primitive leukemia stem cells (LSC). We have reported that SIRT1, a NAD+ dependent deacetylase, is overexpressed in CML LSC and contributes to their maintenance and TKI resistance, related at least in part to increased p53 acetylation and transcriptional activity (Li et.al; Cancer Cell 2012). However, these studies using RNAi and pharmacological inhibitors were limited by possible off-target effects and limited duration of exposure. Therefore, to definitively delineate the role of SIRT1 in CML development, we developed a conditional SIRT1 knockout mouse model by crossing SIRT1 floxed mice (SIRT1fl/fl) with the Mx1-Cre strain to delete SIRT1 in hematopoietic stem cells (HSC), and crossed with transgenic BCR-ABL mice (BA Mx1-Cre SIRT1fl/fl). SIRT1 deletion in normal HSC did not inhibit normal hematopoiesis. In contrast, SIRT1 deletion in LSC profoundly inhibited the development of CML, and SIRT1-deleted CML mice did not develop evidence of morbidity and demonstrated significantly lower leukocytosis, neutrophilia and splenomegaly, and markedly improved survival, compared to controls (Fig-1). CML development in the SCL-tTA/BCR-ABL mouse model is associated with reduction in BM stem cells, and increase in stem/progenitor cell populations in the spleen. Splenic stem and progenitor populations were significantly decreased in SIRT1-deleted mice, whereas BM LTHSC were increased, indicating reversal of alterations associated with CML. SIRT1 deletion was associated with a significant decrease in LTHSC in G0 phase and increase in LTHSC in G1 phase of cell cycle. SIRT1-deleted LTHSCs generated significantly lower WBC counts, donor cell and donor LTHSC engraftment following transplantation into wild-type recipients compared to controls, indicating reduced in vivo repopulating ability. Treatment with the BCR-ABL TKI Nilotinib (50mg/kg/day) or vehicle for 4 weeks resulted in additional suppression of CML hematopoiesis in SIRT1-deleted mice. Gene expression analysis identified significant inhibition of mitochondria and oxidative phosphorylation related genes in SIRT1-deleted CML stem cells compared to controls. We performed extracellular flux analysis of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) using the Seahorse XF analyzer to evaluate mitochondrial respiration and glycolysis in c-Kit+ stem/progenitor cells from SIRT1-deleted and control CML and normal mice. CML LSC demonstrated significantly increased ATP-linked respiration, maximal mitochondrial respiration and mitochondrial reserve capacity compared to normal HSC. SIRT1-deletion resulted in significant reduction in basal and maximal mitochondrial respiration, and in mitochondrial reserve capacity, in CML LSC (Fig-2). In contrast, SIRT1-deletion did not result in significant change in mitochondrial respiration in normal HSC, suggesting that effects of SIRT1 deletion on mitochondrial respiration were specific to CML cells. The SIRT1 substrate PGC-1α is a master regulator of mitochondrial biogenesis and activity. Treatment of CML mice with the PGC-1α inhibitor SR-18292 significantly reduced basal, ATP-linked and maximal mitochondrial respiration. Consistent with results obtained with murine LSC, SIRT1 inhibition with TV39OH resulted in significant reduction in mitochondrial respiration in CML CD34+ cells compared to vehicle controls. Treatment of CML mice with Nilotinib did not significantly alter mitochondrial respiration in LSC. Similarly TKI treatment did not affect mitochondrial respiration in CML CD34+ cells compared to vehicle controls. In conclusion, our studies definitively demonstrate that SIRT1 is required for leukemia development, and reveal its critical role in increased mitochondrial respiration in CML LSC. Increased mitochondrial metabolism is kinase-independent, and is mediated by PGC-1a. SIRT1 inhibition results in enhanced inhibition of TKI-treated LSC. Our studies demonstrate an essential role for SIRT1 in altered mitochondrial metabolism in CML LSC, and support further exploration of SIRT1 pathway inhibition for LSC targeting. Disclosures No relevant conflicts of interest to declare.

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Arsenic trioxide (ATO) mediates PML-RARA (promyelocytic leukemia–retinoic acid receptor-α) oncoprotein degradation via the proteasome pathway and this degradation appears to be critical for achieving cure in acute promyeloytic leukemia (APL). We have previously demonstrated significant micro-environment-mediated drug resistance (EMDR) to ATO in APL. Here we demonstrate that this EMDR could be effectively overcome by combining a proteasome inhibitor (bortezomib) with ATO. A synergistic effect on combining these two agents in vitro was noted in both ATO-sensitive and ATO-resistant APL cell lines. The mechanism of this synergy involved downregulation of the nuclear factor-κB pathway, increase in unfolded protein response (UPR) and an increase in reactive oxygen species generation in the malignant cell. We also noted that PML-RARA oncoprotein is effectively cleared with this combination in spite of proteasome inhibition by bortezomib, and that this clearance is mediated through a p62-dependent autophagy pathway. We further demonstrated that proteasome inhibition along with ATO had an additive effect in inducing autophagy. The beneficial effect of this combination was further validated in an animal model and in an on-going clinical trial. This study raises the potential of a non-myelotoxic proteasome inhibitor replacing anthracyclines in the management of high-risk and relapsed APL.

Introduction:Allogeneic hematopoietic stem cell transplantation (HSCT) cures beta thalassemia major (TM). Such individuals, ex-thalassemics, have good long term survival. However, there is limited data on long term outcome (LTO) of this therapy. This is particularly relevant as these patients often have organ dysfunction pre-transplant due to secondary hemosiderosis apart from the impact of post-transplant factors such as chronic GVHD, chimerism status and iron depletion therapy (IDT). In this report, we describe the LTO of patients with TM who underwent HSCT with busulfan (Bu) and cyclophosphamide (Cy) conditioning at our center from 2000 to 2011 and had a minimum of 2 year follow-up. Method: Data was extracted from prospectively maintained standardized case record forms for details of HSCT and long term follow-up with particular reference to GVHD, chimerism (evaluated at day +30, +60, +100 and thereafter as indicated), IDT (initiated at variable periods post-HSCT) and metabolic and endocrine disorders evaluated on physician discretion or as per clinical indications. Results:A total of 190 patients underwent matched related donor HSCT from 2001 to 2011 with Bu/Cy based conditioning. After excluding those who expired or had primary graft failure or did not have at least 2 years of follow-up, 124 patients were available for analysis of LTO. 44 patients (35.5%) class 3, 69 patients (55.6%) class 2 and 11 patients (8.9%) class 1. The median age was 7 years (range: 2-24) with 81 males (65.3%). The median follow-up was 7 years (range: 2 to 14). Chronic GVHD was present in 22 patients (17.7%]. Mixed chimerism (MC) occurred in 40 patients (32%) in the first year after HSCT: level I in 21 (52.5%), level II in 10 (25%), level III in 7 (17.5%), and level unknown in 2(5%). At last follow-up, 20/40 (50%) patients with MC went on to CC, 18 maintained stable MC (level I-5, level II-9 and level III-4) with hemoglobin of 11.35g/dl (range: 9-13.5), while 2 (5%) with level 3 MC remained transfusion dependent. Median serum ferritin (SF) at HSCT was 2367 ng/ml (range: 685-7660). IDT was initiated in 90 (72.6%) patients at a median of 15 months (range: 6-53) post-HSCT - 13 patients (14.4%) were treated with phlebotomy alone, while 39 (43.3%) received chelation and 38 (42.2%) the combination. Reduction in SF/month [absolute quantity (ng/ml/month) and percent] was as follows: 40.5 (range: 11.68 - 125.78); 1.67% (range: 0.5-4.58), 54.9 (range: 9.3- 278.7); 2.1% (range: 0.41- 13.8) and 36.6 (range: 3.51-590.7); 1.3% (range: 0.42-42.99), in the phlebotomy, chelation and combination groups, (p=0.077 & 0.017, respectively). SF level of Conclusion: Our data shows that even though the long term survival of ex-thalassemics is extremely good, at least 40% of them suffer from several co-morbidities related to iron overload and various metabolic and endocrine disorders which requires a coordinated plan for their management. The aim therefore should be to transplant these patients as early as possible before such complications occur and implement IDT intensively early after HSCT. Disclosures No relevant conflicts of interest to declare.

Despite the success of tyrosine kinase inhibitors (TKIs) in treatment of CML, cures remain elusive, as primitive leukemia stem cells (LSC) are retained in patients achieving remission. Previous studies from our group have suggested that Sirtuin 1 (SIRT1) inhibition may represent a novel approach for elimination of LSCs in chronic phase CML. SIRT1 was shown to be overexpressed in CML LSCs, and SIRT1 inhibition using shRNA or a small molecule SIRT1 inhibitor selectively eliminated CML LSCs by increasing p53 acetylation and activity (Li et.al; Cancer Cell 2012). These studies were limited by possible off-target effects and limited duration of in vivo exposure. Here we used a genetic mouse model to definitively delineate the role of SIRT1 in CML development. A model for conditional SIRT1 deletion in hematopoietic stem cells was established by crossing homozygous SIRT1 exon-4 floxed (SIRT1fl/fl) mice with Mx1-Cre mice. To study the requirement of SIRT1 for development of CML, Mx1-cre SIRT1fl/fl mice were crossed with SCL-tTA/BCR-ABL mice, representing a tet-regulated inducible transgenic mouse model of CML, to generate SCL-tTA/BCR-ABL Mx1-Cre SIRT1fl/fl mice (BA Mx1-Cre SIRT1fl/fl). BA SIRT1fl/fl mice lacking Mx1-Cre were used as controls. The mice were maintained on doxycycline until CML induction. Cre mediated deletion of SIRT1 was induced by intraperitoneal pIpC injections (250µg/mouse) administered every other day for a total of 7 doses. SIRT1 knockdown was confirmed by PCR for excised exon-4 and by RT-Q-PCR. Bone marrow (BM) cells from either BA Mx1-Cre SIRT1fl/fl or controls (both CD45.2) were transplanted into irradiated (800 cGy) CD45.1 congenic recipients (2X106 cells/mouse). Cre-mediated deletion of SIRT1 was induced by pIpC injection starting at 4 weeks post-transplant, followed by withdrawal of tetracycline to induce BCR-ABL expression. Serial PB counts and phenotypic evaluation of cell types by flow cytometry (Fig 1 A-B) showed SIRT1 knockdown to have a profound effect on CML development. By 8 weeks after BCR-ABL induction, BA SIRT1fl/fl mice (n=10), showed significantly lower neutrophils (p=0.0003) and Gr-1/Mac-1 positive myeloid cells (p=0.0002) compared to control mice. Subsequently, control mice developed progressive neutrophilic leukocytosis and increasing morbidity from leukemia, whereas BA SIRT1fl/fl mice demonstrated significantly lower WBC counts, without evidence of progressive increase or morbidity (Fig 1 A). This cohort of mice continues to be followed for survival. Another cohort of BA Mx1-Cre SIRT1fl/fl mice was sacrificed at 8 weeks post pIpC injection and BCR-ABL induction to evaluate the effect of SIRT1 knockdown on stem and progenitor populations (n=6 each). SIRT1 deleted mice demonstrated significant reduction in spleen size, weight, cellularity, and myeloid infiltration (Fig 2 A-B), and in myeloid cell expansion in the BM compared to controls (p=0.002). Primitive lineage negative, Sca1 positive, c-Kit negative (LSK) cells and granulocyte-macrophage progenitors (GMP) were significantly reduced in BM and spleen of BA SIRT1 deletedmice compared to control mice, whereas megakaryocyte-erythrocyte progenitors (MEP) were increased (Fig 3 A-B). Long term hematopoietic stem cells (LTHSC) in the BM are reduced following CML development. The percentage and number of LTHSC were significantly increased in SIRT1 deletedmice compared to control mice (Fig 3C-D). We also evaluated the effect of SIRT1 deletion on normal hematopoiesis by studying Mx1-Cre SIRT1fl/fl mice lacking BCR-ABL. SIRT1fl/fl mice without Mx1-Cre were studied as controls. Mx1-Cre SIRT1fl/fl and control mice were treated with pIpC to induce SIRT1 deletion. SIRT1deletedmice did not show significant alteration in blood counts, but demonstrated significantly higher LSK and LTHSC numbers in BM compared to control mice. Upon secondary transfer, recipients of BM from SIRT1deleted mice showed a modest increase in donor cell engraftment at 12 weeks compared to controls (90.8% (83.2-92.2%) vs 83.6% (75.8-86.7%); p=0.001). We conclude that genetic deletion of SIRT1 markedly inhibits all aspects of CML development in transgenic BCR-ABL mice, without impairing normal hematopoiesis. These observations demonstrate a critical role for SIRT1 in leukemia development, and support further evaluation of SIRT1 as a therapeutic target in CML. Disclosures No relevant conflicts of interest to declare.

Aim: Cytidine deaminase (CDA) irreversibly deaminates cytarabine (Ara-C), a key component of acute myeloid leukemia (AML) induction and consolidation therapy. CDA overexpression results in Ara-C resistance, while decreased expression is associated with toxicity. We evaluated factors influencing variation in CDA mRNA expression in adult AML patients and normal controls, and how they contributed to Ara-C cytotoxicity in AML cells. Materials & methods: CDA mRNA expression in 100 de novo AML patients and 36 normal controls were determined using quantitative reverse-transcriptase PCR. Genetic variants in the CDA gene were screened by direct sequencing. IC50 of Ara-C was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Results: CDA RNA expression as well as Ara-C IC50 showed wide variation in AML samples and normal controls. Fourteen sequence variants were identified, three of which (-33delC, intron 2 TCAT repeat and the 3´untranslated region 816delC variants) showed significant association with RNA expression and the nonsynonymous coding variant 79A>C was associated with Ara-C cytotoxicity. Conclusion: CDA genetic variants explain the variation in RNA expression and may be candidates for individualizing Ara-C therapy. Original submitted 8 July 2011; Revision submitted 10 October 2011

To cite this article: Sumitha E, Jayandharan GR, David S, Jacob RR, Sankari Devi G, Bargavi B, Shenbagapriya S, Nair SC, Abraham A, George B, Viswabandya A, Mathews V, Chandy M, Srivastava A. Molecular basis of Bernard–Soulier syndrome in 27 patients from India. J Thromb Haemost 2011; 9: 1590–8. Summary. Background: Bernard–Soulier syndrome (BSS) is an extremely rare (1:1 million) bleeding disorder of platelet adhesion, caused by defects in the glycoprotein (GP)Ib/IX/V complex. Patients and methods: The diagnosis in 27 patients was based on low platelet count, presence of giant platelets and aggregometry studies. Flow cytometry to assess the surface GPIb/IX/V complex showed reduced (7.7–57%) expression. gDNA was screened for mutations in the GPIBA, GPIBB, GP9 genes using PCR-conformation sensitive gel electrophoresis (CSGE). Results: Thirteen different disease-causing mutations, including missense (54%), frameshifts (38%) and nonsense (8%) mutations, were identified in 27 patients. Eleven of them were novel including five novel frameshifts (GPIbα: p.Gln97_98fsX113, p.Pro402_403fsX52; GPIbβ: p.Arg17fsX14; GPIX: p.Gly24fsX43, p. Pro130fs, a nonsense mutation (GPIX, p.94, Gln>X) and five novel missense mutations (GPIbα: p.492, Tyr>His; GPIbβ: p.65, Pro>Arg, p.129, Gln>His, p.132, Leu>Pro; GPIX: p.55, Phe>Cys). Interestingly, four common mutations, Cys8Arg (n = 6) and Phe55Ser (n = 2), Phe55Cys (n = 2) in GPIX and a novel 22-bp deletion in the GPIBB gene predicting p.Arg17fsX 14 (n = 10) were seen in 20 patients. Conclusion: The molecular data presented here is the largest series of BSS patients to be reported so far, adding significantly to the mutation database of this condition and also useful for its genetic diagnosis in India.

A toxicity reduced conditioning regimen containing Treosulfan (Treo), fludarabine (Flu), thiotepa for high risk Thal Major (TM) has been used since 2009 at our centre that has significantly improved transplant outcomes of these patients compared to the historical cohort of patients receiving busulfan/ cyclophosphamide based myeloablative regimen (Mathews et al, 2013). Limited knowledge is available on the pharmacokinetics (PK), pharmacogenetics (PG) and pharmacodynamics of fludarabine and treosulfan, especially in non-malignant hematological disorders like TM. We describe here the PK of Flu and Treo in patients with TM undergoing HSCT, the factors influencing the inter-individual variability in PK and the role of these factors on HSCT outcome. Seventy one patients diagnosed with TM undergoing HSCT with Flu/Treo based conditioning regimen between January 2012 and January 2015 were included (Table: Patient demographics). Selected functional polymorphisms in the NT5E, DCK, hENT1 and GST genes that are involved in fludarabine or treosulfan metabolism were screened. All patients received Flu 40mg/m2/day x 4 days as an 1hr infusion on days 1 and 4 and Treo as 14g/m2/day x 3 days at the rate 5g/hr. Plasma was separated from the peripheral blood collected at predetermined time points after the infusion of Flu and Treo PK analysis. Plasma Flu was analyzed using a LC-MS/MS method and the concentration was expressed as mMole/ml while Treo was analyzed using a HPLC-RI method and concentration was expressed as mg/L. Flu and Treo PK was estimated using nonlinear mixed effects modeling via Monolix 4.3.3. The covariates tested for both PK were: age, sex, body weight, BSA, ferritin, and polymorphisms in NT5E, hENT1, dCK and GST genes. The PK parameters AUC, CL, V and k were estimated on day 1 for Treo and on day 1 and day 4 for Flu (Table). The influence of Flu and Treo PK and PG on graft rejection, early transplant related mortality (TRM) & chimerism status was estimated using logistic regression analysis. Wide inter-individual variation in Flu and Treo PK was noted (7 and 9 fold Vs 5 and 8 fold respectively for Day 1 & 4 Flu AUC & Cl; 33 & 31 fold variation in Treo AUC and Cl) (Table). Flu CL was significantly higher on day 4 compared to day1 (Figure A). The variation in Flu PK was explained by genetic variants in NT5E and dCK. Patients having variant genotype for the SNPs in NT5E (rs2295890) and dCK (rs11544786) showed significantly lower plasma Flu clearance compared to those with wild type genotype (p=0.006 & p=0.05 respectively) (Figure B). This is consistent with our previous report in patients with aplastic anemia undergoing HSCT (Mohanan et al. 2014; Blood: 124 (21)). None of the genetic variants in the GST genes explained the variation in Treo PK. Day21 mortality was seen in 6/71 patients (8.5%) and graft rejection in 3/66 evaluable patients (4.5%). Analysis of the influence of PK and PG variables on transplant outcome showed significantly high first dose Flu AUC to be associated with D21 mortality upon Univariate analysis (median 42.5, range 32.1-63.7 compared to 31.8, range 15.2-111 mMole*h/mL, in those with and without TRM respectively; p=0.043); none of these parameters were significantly associated with graft rejection or mixed chimerism. There was no association between Treo PK parameters and graft rejection or TRM. The influence of Flu and Treo PK on regimen related toxicity is yet to be evaluated. The lack of the influence of PK on transplant outcome could be due to lower incidence of rejection and TRM in this cohort. Further analysis in a larger cohort of patients will be done once we enroll more patients for PK analysis. Our results demonstrate that Flu PK is influenced by genetic variants in NT5E and dCK, the enzymes involved in Flu biotransformation. The relationship between high-plasma Flu exposure and TRM and given the fact that multiple factors influence TRM, we can extrapolate that the plasma Flu AUC may be a surrogate marker of overall preparative regimen intensity as reported previously (Long-Boyle et al, Bone Marrow Transplant, 2011). The lack of association of genetic variants in GST genes in explaining the inter-patient variability in treosulfan exposure suggests the involvement of other drug metabolizing genes on treosulfan PK. We are currently evaluating the role of genetic variants in a large panel of drug metabolizing genes on explaining this inter-individual variability in Treo PK. Disclosures No relevant conflicts of interest to declare.

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.

Daunorubicin (Dnr) has been the choice of anthracycline in the induction chemotherapy of Acute Myeloid Leukemia (AML) along with Cytarabine for the past 4 decades. Drug resistance and relapse of the disease has been the major concern in the success of AML therapy. Several factors, including pharmacokinetics (PK) of Dnr could have impact on treatment outcome as pharmacokinetic effects might limit the amount of drug that reaches the tumor. In this study, we analyzed the factors influencing plasma PK of Daunorubicin and its major metabolite Daunorubicinol (DOL) in AML patients receiving induction chemotherapy and compared PK parameters with clinical outcome (relapse). Sixty four adult denovo AML patients were enrolled in the study after obtaining informed consent. Dnr was given as 1 hour bolus infusion at a dose of 60mg/m2/day for the first 3 days of induction chemotherapy. Peripheral blood were collected at 0, 15min, 1hr, 2hr, 4hr, 6hr and 24hr on the first day of induction chemotherapy. Plasma levels of Dnr and DOL were measured using high performance liquid chromatography coupled with fluorescence detector. Population PK was analyzed by nonlinear mixed effects modeling using Monolix version 4.3.2. Common single nucleotide polymorphisms in ABCB1, ABCG2, CBR1 and CBR3 genes were screened by PCR followed by either sequencing or restriction digestion. In vitro cytotoxicity of the primary AML cells to Dnr was assessed by MTT cell viability assay and IC50 was calculated using ADAPT5 software. The role of plasma Dnr PK on relapse of disease was estimated using nonparametric Mann-Whitney U test. Demographics of the patients enrolled in the study are listed in table 1. PK parameters of Dnr and DOL showed wide inter-individual variation (table2). Older patients had a trend towards lower Dnr clearance (CL) (p=0.09). Patients harboring FLT3-ITD mutations had higher CL than the patients without mutation though not reaching statistical significance. Patients with variants of SNPs rs20572 (exon3) and rs9024 (3’UTR) in CBR1 (which were in complete linkage disequilibrium) had a trend towards significantly higher Dnr AUC [median-0.2711; (0.04903-2.165) vs. median 0.2117; (0.0546-0.9036); p=0.06] than patients with wild-type. Among the 64 patients, complete remission (CR) was documented in 42 (65%) of which, 31% relapsed eventually [median time to relapse 328 (104-864) days]. AML patients who relapsed had significantly lower Dnr AUC [median 0.17 (0.049-2.165) vs. median 0.31 (0.1-1.74); p=0.009] and higher DOL AUC [median 8.67 (1.6-20.1) vs.4.99 (1.3-16.5); p=0.015].Also, relapsed patients had higher Dnr clearance (CL)[median -546.54 (41.5-1632)vs. 329.3 (42.9-944.5); p=0.011] and lower DOL CL[median-9.8 (4.9-56)vs. median- 19.7 (6.1-57); p=0.015]than patients who did not relapse. IC50 of Dnr was higher in patients who relapsed (median-0.7 (0.02-1.46) vs. median- 0.23 (0.007-1.11); p=0.056) than in patients who did not relapse. To the best of our knowledge, this is the first population PK report in AML which is designed to evaluate various factors which might influence Dnr and DOL PK in AML. Though the number is small, our results suggest that plasma PK of Dnr influences clinical outcome in AML. Lower systemic exposure of Dnr in relapsed patients suggests that amount of drug required to completely eradicate the leukemic cells is not achieved and escalating dose might be an option to decrease relapse rates. Cellular pharmacokinetics of Dnr in these patients is ongoing in the lab, which will be more informative and help in prognosticating and dose adjustment for better treatment outcome. This data provides a probable biological basis for the previously reported clinical benefit of decreased relapses following the increase of the dose of DNR in induction to 90mg/m2/day (Tallman et al. NEJM 2009).Wide inter-individual variation in Dnr PK also suggests that this increased dose may not be necessary for all patients. Further studies are required to confirm this finding in a larger cohort of patients and to work towards strategies for TDM of Dnr in AML Figure 1 Figure 1. Figure 2 Figure 2. Disclosures Srivastava: Octapharma: Consultancy, Other.

Acute myeloid leukemia (AML) is a clinically and biologically complex and heterogeneous hematopoietic neoplasm. Recent advances in acute myeloid leukemia (AML) biology have lead to prognosticate and predict treatment outcome in AML based on molecular markers. Mutations in NPM1, CEBPA are considered good prognosis and High BAALC, ERG & MN1 expression associate with worse outcome in AML patients treated with standard chemotherapy. Although many efforts have been made to identify genetic mutations and modulated gene expression levels that can be used to predict outcomes in patients with AML, the association between these prognostic markers has not been evaluated. We have reported previously that the NPM1 mutated patients have significantly high dCK and hENT1 gene expression (involved in cytarabine metabolism) and low ABCG2 and ABCB1transporter expression (Abraham et al, ASH abstracts; Nov 2011; 118: 3481 and Nov 2012; 120: 143), suggesting that the good prognostic nature of this mutation is possibly due to the better metabolism and transport of the chemotherapeutic drugs used in induction therapy. We extended this study to look for association between NPM1/FLT3 mutation status and the RNA expression of other good or poor prognostic markers in patients with AML. We prospectively included 274 adult patients with AML in this study. The median age was 42 years (range 16-74y). AML was diagnosed according to the FAB and WHO classifications. There were 238 patients with de novo AML; Secondary AML -6; Therapy related AML- 2 and Relapsed AML-28. Bone marrow cytogenetics and immunophenotyping analysis was available for all patients at diagnosis and/or relapse. Diagnostic bone marrow MNCs were isolated by ficoll- density gradient centrifugation and stored in trizol reagent for RNA expression and mutation detection. RT-PCR was used to screen AML-ETO and Inv 16, and the expression of BAALC, ERG1, MN1, CXCR4 and WT1were analyzed using RQ-PCR. NPM1-c, FLT3 ITD and TKD were screened using DNA PCR followed by gene-scan, sequencing or RFLP methods. The basic demographics and the frequency of the markers are listed in Table 1. When analyzed separately in normal karyotype AML (NK-AML), the frequencies of the mutations were: NPM1: 52.2%; FLT3-ITD: 24%; TKD: 4.3%. When the RNA expression of BAALC, WT1, ERG1, CXCR4 and MN1 was compared in patients with NPM1 or FLT3 mutations, we noticed that patients with NPM1 had significantly low expression of BAALC, MN1 and ERG1 while those with FLT3 mutations (ITD or TKD) had higher expression of these genes (Figure1). There was no significant association with CXCR4 or WT1 expression and these mutations. When analyzed separately in the normal karyotype AML, these associations were still significant. In addition, the relapsed patients had significantly higher expression of BAALC, MN1, and ERG1 RNA compared to de-novoAML cases (data not shown). To conclude, we show that NPM1 or FLT3 mutations acquire the prognostic significance due to several factors including BAALC, ERG1 and MN1expression levels in addition to drug metabolizing enzymes’ and drug transporter expression. These factors must be taken into consideration when attempting to personalize chemotherapy in AML. Disclosures: No relevant conflicts of interest to declare.

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. 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 (IC506.25µM for Ara-C;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 ( 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. Disclosures: No relevant conflicts of interest to declare.

Wide inter-individual variation in terms of treatment outcome and toxic side effects of treatment exist among patients with AML receiving chemotherapy with cytarabine (Ara-C) and daunorubicin. We have previously evaluated the expression of the major genes involved in cytarabine transport and metabolism on ex-vivo Ara-C response and compared it with cytogenetic and molecular markers in AML (Blood (ASH Annual Meeting Abstracts) 2011 118: Abstract 3481). Our candidate gene expression data led us to propose Ara-C resistance index (Ara-C RI) (RI = ΔCT (DCK X ENT1)/ ΔCT CDA), which incorporates candidate Ara-C metabolizing genes whose RNA expression are significantly associated with ex-vivo Ara-C cytotoxicity. Ara-C RI values were significantly higher in resistant (IC50 >80 uM) and intermediate (IC50 6.25-80uM) samples when compared to sensitive samples (IC50 Though, the Ara-C RI correlated well with ex-vivo cytotoxicity as well as treatment response in vivo (data not shown), this did not completely explain the variation, suggestive of alternative resistance mechanisms. We undertook a genome-wide gene expression profiling to address possible mechanisms of Ara-C resistance other than the candidate gene approach. Based on ex vivo Ara-C cytotoxicity at diagnosis, Ara-C sensitive (IC50 80uM) (each 5) were included for microarray analysis. Total RNA was extracted from bone marrow mononuclear cells using tri reagent, cDNA was synthesized and then subjected to one color 8 X 60K Agilent microarray analysis. Data was normalized, filtered and analyzed using Gene Spring GX (V 12.0) software. Normalization was done using the 75th percentile shift (Percentile shift normalization is a global normalization, where the locations of all the spot intensities in an array are adjusted). Using unpaired t-Test, 4436 genes were identified to be differentially expressed (Fold change expression values were provided as log-base 2) between Ara-C sensitive samples and Ara-C resistant samples. Differentially regulated genes were clustered using hierarchical clustering based on Pearson coefficient correlation algorithm to identify significant gene expression patterns. Genes were classified based on functional category and pathways using GeneSpring GX and Genotypic Biointerpreter-Biological Analysis Software. The differentially expressed genes fell into the following biological processes; transcription (375 genes), transport (364 genes), metabolism (267 genes; Fig 1d), immune (155 genes), cell cycle (129 genes), apoptosis (123 gene; Fig 1c) and so on. Upregulated gene list in Ara-C sensitive group included apoptotic related genes like PMAIP1, NDUFA, BAX, BCL2, TRAF2, transcriptional regulators including SMAD5, SMAD1, ZNF family proteins- ZNF644, ZNF469, ZNF195, ZNF22, ZNF3, ZNF713, ZNF777, ZNF234, CEBPa, PARP, ETV6, E2F3 and so on. Down-regulated genes in Ara-C sensitive group are of special interest as they could be potential candidates for targeting Ara-C non-responsive group. They included transcriptional regulators like HDAC4, KLF4, CREB5, CEBPb, RARA, E2F4, MNDA, MTA3 and PPARd also apoptotic genes like MCL1, PSEN1, ELMO2, PAK1, APAF1, MAPK1, CD40, FAS, CASP1 and CASP8. Interestingly, many of the genes involved in cellular metabolism were found to be down regulated in AraC sensitive group. Major down-regulated metabolic genes include CDA, SLC2A3, SLC2A8, GK, NADK, ACACB, ACSL1, PFKFB4, PFKFB3, PDK4, ME1 and PC. This study has come up with previously unrecognized aspects in Ara-C resistance in AML, and is suggestive of Ara-C sensitive samples having increased expression of anti-Warburg set of genes. Our data as well as growing evidences from various malignancies and altered cellular metabolism propose the possibility of using metabolic inhibitors alone or in combination with Ara-C to overcome drug resistance. Disclosures: No relevant conflicts of interest to declare.

Abstract 1438 Combination chemotherapy in Acute Myeloid Leukemia (AML) can cure approximately 25% of patients but the majority is still non-responsive. Drug resistance and relapse are considered to be the major reasons for treatment failure. Though overexpression of ATP-Binding-Cassette (ABC) transporters including ABCB1 and ABCG2 have been shown associated with lower remission rates and survival in AML, the role of majority of ABC transporter genes are still unknown. Present study aims to determine the role of candidate ABC transporter RNA expression (that are shown to directly or indirectly influence sensitivity towards Cytarabine (Ara-C) or Daunorubicin (Dnr)), on in vitro cytotoxicity and the influence of prognostic markers (NPM, FLT3-ITD, AML-ETO, CBFB-MYH11 and CD34) on these transporter expression. 177 adult AML patients (median age: 43 years (16–74 years)) other than AML-M3 were included in this study. Total RNA was extracted from diagnostic bone marrow mononuclear cells and expression levels for each target gene (ABCB1, ABCB6, ABCB10, ABCA3, ABCA5, ABCA6, ABCC3, ABCC11 and ABCG2) relative to housekeeping gene GAPDH was analyzed using Taqman based gene expression assays. ABCB6 and ABCC3 assays were custom designed and were normalized to ABL as housekeeping gene. The dCT values (where dCT = CT value of target gene – CT value of housekeeping gene; lower dCT corresponds to higher expression and vice versa) were used for comparing expression between groups. In vitro cytotoxicity was assessed using MTT cell viability assay and IC-50 was calculated. Based on Ara-C and Dnr IC50, these samples were categorized as sensitive or resistant (IC50 5.2 uM for Ara-C; 0.5uM for Dnr). FLT3-ITD and NPM mutation status at diagnosis were determined by PCR followed by Genescan analysis using genomic DNA samples. AML-ETO and CBFB-MYH11 status were determined by RT-PCR. Wide variation in ABC transporter RNA expression was seen among AML patients (Fig 1a). Both ABCC3 and ABCB6 RNA expression were significantly higher (median ABCC3 dCT= 7.68 (−1.80–15.97) vs 10.62 (−0.66– 17.77); p= 0.0005; median ABCB6 dCT= 5.0 (−0.28 – 8.25) vs 5.4 (1.76 – 12.17); p=0.03)) in Dnr resistant patients (n=65) when compared to those who are sensitive to Dnr (n=53) (Fig 1b). No association was seen between any of these transporter expression and Ara-C in vitro cytotoxicity. Effects of NPM and FLT3 mutation status on ABC transporter genes were then evaluated. ABCB1 expression was significantly lower and ABCA5 expression was higher in patients with NPM mutation (n=50) when compared to those without mutation (n=126)-Table1. Patients with FLT3-ITD mutation (n=26) had a significantly lower ABCB1 expression when compared to those without mutation (n=150)-Table1. Low ABCB1 in this group could probably be due to the fact that NPM mutation was also positive in 16 out of 26 FlT3-ITD mutated patients. ABCB6 expression was significantly lower in FLT3-ITD positive patients (Table1). Other mitochondrial transporters like ABCG2 and ABCB10 showed a trend to low expression in FLT3-ITD patients when compared to those without FLT3 mutation (p=0.06 and p=0.1 respectively, Table1). Eighteen patients were positive for AML-ETO (n=12) or CBFB-MYH11 (n=6) and the RNA expression of ABCA5, ABCA3 and ABCG2 were significantly lower in this group compared to those who were negative for AML-ETO or CBFB-MYH11 (Table1). In addition, ABCB1 and ABCG2 expression was significantly higher in CD34 positive patients (CD34 >20% as analysed by flow cytometry) when compared to those with low CD34 expression (Table1) This study has come up with previously unrecognized aspects in ABC transporter expression and its role in AML. Low expression of ABCB1, a well accepted candidate for drug resistance, in NPM mutated AML could be one of the reasons which make NPM mutation a favourable prognostic marker. ABCC3 and ABCB6 which have shown association with Dnr sensitivity in this study could be a key avenue for further investigation. This comprehensive analysis on ABC transporter expression and its association with in vitro cytotoxicity and prognostic markers suggests ABCC3, ABCB6 and ABCA5 as probable targets which can be modulated for improving chemotherapeutic responses. Disclosures: No relevant conflicts of interest to declare.

Abstract 2484 Treatment failure in AML can be attributed to resistance to chemotherapeutic drugs. Induction chemotherapy of AML involves combination of Daunorubicin (Dnr) and Cytarabine. Up-regulation in the expression of efflux transporters such as ABCB1 and ABCG2 has been shown to be one of the causes of Dnr resistance in AML. Apart from efflux transporters, increased expression of the Dnr metabolising enzymes (Carbonyl reductase 1 (CBR1) and CBR3) also can influence the cytotoxic activity of Dnr against leukemic cells. Aim of the present study is to investigate: i) the role of mRNA expression of Dnr metabolising enzymes (CBR1 and CBR3) on the in vitro cytotoxicity of Dnr in primary AML cells and ii) effect of Single nucleotide polymorphisms (SNPs) in CBR1 and CBR3 on the plasma pharmacokinetics of Dnr and Daunorubicinol (DOL) in AML patients. Ninety patients with AML (excluding AML-M3) at diagnosis before the initiation of therapy were included. Bone marrow (BM) was collected at diagnosis and blast percentage in our cohort of patients ranged from 20–100%. Mononuclear cells (MNCs) were isolated by density gradient centrifugation. Total RNA was extracted from BMMNCs and cDNA was prepared. CBR1 and CBR3 mRNA expression was measured using Taqman based quantitative RT-PCR normalized to GAPDH. In-vitro cytotoxicity of Dnr was determined by MTT cell viability assay. Intracellular levels of Dnr and DOL was estimated by HPLC coupled with Fluorescence detector (FD). SNPs in CBR1 and CBR3 were screened by PCR followed by sequencing. In addition, plasma samples during Dnr infusion was collected at 0, 1, 2, 4, 6 and 24hrs from 24 of the 90 AML patients. Levels of Dnr and DOL in the plasma samples were estimated by HPLC coupled with FD and Area under Curve (AUC) for Dnr and DOL were calculated. CBR1 (range: 0.02–253.15) and CBR3 (range: 8.24– 29296.67) mRNA expression showed wide inter-individual variation. Intracellular levels of Dnr (range: 17.98–1718ng/3×106cells) and DOL (range: 2.76–62.07ng/3×106cells) showed 95 and 22 fold variation respectively. IC50 of Dnr in these samples ranged from 0.05 to 3.15 μM. Based on the IC50, samples were categorized as sensitive (IC501.0μM) to Dnr. CBR1 expression was significantly higher in resistant samples (median-102.99: range: 7.18–253.15) compared to intermediate (median- 29.73: range: 0.02–224.23) and sensitive samples (median-18.67: range: 0.34–105.40 p=0.0154) [Figure 1]. In addition, significant correlation between CBR1 expression and intracellular DOL levels was observed (r= 0.52; p Five SNPs in CBR1 and 3 in CBR3 were identified. CBR1 SNPs, rs20572, rs9024 and rs25678 were in complete linkage disequilibrium (LD). Variant of rs25678 and rs20572/rs9024 had an allelic frequency of 0.244 and 0.20 respectively. Among CBR3 SNPs, variants of two non-synonymous polymorphisms rs8133502 and rs1056892 showed allelic frequency of 0.31 and 0.51 respectively. AUC of Dnr and DOL in plasma samples ranged from 22–2474ng*h/ml and 37–2027 ng*h/ml respectively while clearance of Dnr ranged from 0.04 to 3.34 L/h. Plasma DOL AUC, unlike in vitro intracellular DOL, did not show any significant association with CBR1 and CBR3 expression. In patients with variant rs25678 genotype, Dnr AUC was higher and clearance was lower compared to the wild type genotype, reaching a trend to statistical significance (p=0.0537 and 0.1184 for Dnr AUC and clearance). Influence of Dnr and DOL plasma levels on clinical outcome, if any, remains to be evaluated. This is the first report showing the influence of CBR1 and CBR3 in in-vitro cytotoxicity of Dnr and role of SNPs in CBR1 and CBR3 in plasma pharmacokinetics of Dnr in AML patients. To conclude, higher expression of CBR1 confers resistance to Dnr in primary AML cells and polymorphisms of this gene may influence the pharmacokinetics of Dnr. Disclosures: No relevant conflicts of interest to declare.