Myelogenous Leukemia Patients Research Articles

A Concise Review on Analytical Methods for Determination of Nilotinib

Abstract: Nilotinib hydrochloride is a tyrosine kinase inhibitor licensed to treat chronic myelogenous leukemia in patients with the Philadelphia Chromosome (Ph+). Researchers at Novartis Pharmaceuticals discovered novel inhibitors that are effective against imatinib-resistant BCR-ABL mutations. As a consequence, Nilotinib was discovered. Several analytical approaches were employed to address the quantitative as well as qualitative assessment of Nilotinib from diverse biological and pharmaceutical matrices during the development of Nilotinib. The literature search was conducted by evaluating publications reporting on nilotinib analytical methodologies from 2006 to 2022. This review briefly summarizes the drug profile, viz. stereochemistry, mechanism of action, resistance, pharmacokinetics, pharmacodynamics, side effects, and several analytical techniques used to assess Nilotinib in dosage form, bulk, and biological fluids. The determination of Nilotinib using analytical methods is important for therapeutic drug monitoring, optimizing dosage, ensuring safety and efficacy, and conducting comparative studies. A variety of techniques are gathered and examined, including spectroscopy, electrophoresis, voltammetry, Raman spectroscopy, differential scanning calorimetry, X-ray diffraction, chromatography, and hybrid techniques. They are also useful for studying the pharmacokinetics of the drug. These methods play a crucial role in the effective and personalized treatment of patients with chronic myeloid leukemia and other conditions where Nilotinib is used.

FLT3-ITD transduces autonomous growth signals during its biosynthetic trafficking in acute myelogenous leukemia cells

FMS-like tyrosine kinase 3 (FLT3) in hematopoietic cells binds to its ligand at the plasma membrane (PM), then transduces growth signals. FLT3 gene alterations that lead the kinase to assume its permanently active form, such as internal tandem duplication (ITD) and D835Y substitution, are found in 30–40% of acute myelogenous leukemia (AML) patients. Thus, drugs for molecular targeting of FLT3 mutants have been developed for the treatment of AML. Several groups have reported that compared with wild-type FLT3 (FLT3-wt), FLT3 mutants are retained in organelles, resulting in low levels of PM localization of the receptor. However, the precise subcellular localization of mutant FLT3 remains unclear, and the relationship between oncogenic signaling and the mislocalization is not completely understood. In this study, we show that in cell lines established from leukemia patients, endogenous FLT3-ITD but not FLT3-wt clearly accumulates in the perinuclear region. Our co-immunofluorescence assays demonstrate that Golgi markers are co-localized with the perinuclear region, indicating that FLT3-ITD mainly localizes to the Golgi region in AML cells. FLT3-ITD biosynthetically traffics to the Golgi apparatus and remains there in a manner dependent on its tyrosine kinase activity. Tyrosine kinase inhibitors, such as quizartinib (AC220) and midostaurin (PKC412), markedly decrease FLT3-ITD retention and increase PM levels of the mutant. FLT3-ITD activates downstream in the endoplasmic reticulum (ER) and the Golgi apparatus during its biosynthetic trafficking. Results of our trafficking inhibitor treatment assays show that FLT3-ITD in the ER activates STAT5, whereas that in the Golgi can cause the activation of AKT and ERK. We provide evidence that FLT3-ITD signals from the early secretory compartments before reaching the PM in AML cells.

Abstract 738: Comparative Cardiotoxicity of Tyrosine Kinase Inhibitors Ponatinib and PF114

Background: The advent of tyrosine kinase inhibitor (TKI) targeted therapy revolutionized the treatment of chronic myelogenous leukemia (CML) patients, leading to a significant impact on remission rates and overall survival. However, cardiotoxicity associated with these targeted therapies put the cancer survivors at greater risk. Ponatinib is an example shown unexpected wide spectrum of cardiotoxic complications which limits its clinical applications. A newly designed 4 th -generation tyrosine kinase inhibitor PF114 having better target spectra in CML, could be a potential alternative to ponatinib. But the cardiotoxic potential of PF114 is not yet fully understood. Objectives: The objectives of the present study were to compare cardiotoxicity of ponatinib with PF114. Methods: In this study, we employed zebrafish transgenic BNP reporter line that expresses luciferase under control of the nppb promoter ( nppb : F-Luciferase) to compare cardiotoxic potential of TKIs. We also exploited NRVMs to explore cardiotoxic mechanism associated with ponatinib and PF114. Results: We observed that increasing dose of PF114 showed minimal induction of the BNP reporter compared to ponatinib. PF114 did not reduce the ventricular fractional shortening in zebrafish as compared to ponatinib. Consistently, in cultured rat cardiomyocytes, PF114 could not reiterate the same cardiotoxic effect of ponatinib as depicted by cell viability and TUNEL assay. Mechanistically, PF114 has shown no effects on essential prosurvival AKT and ERK signaling pathway while ponatinib is known to exerts its cardiotoxic effects by their inhibition. Additionally, the allosteric tyrosine kinase inhibitor ABL001(Asciminib) in combination with PF114 depicts less pronounced cardiotoxic effects than ponatinib in similar settings. Conclusions: This study provides further rationale to utilize zebrafish for the prediction of cardiotoxicity of anticancer drugs. The comparative cardiotoxicity of ponatinib and PF114 suggest that PF114 is a significantly less cardiotoxic analogue and safer treatment option than ponatinib for CML patients harboring BCR/ABL-T315I “gatekeeper” mutation.

Certain Killer immunoglobulin like receptor (KIR)/ KIR HLA class I ligand genotypes influence NK antitumor activity in acute myelogenous leukemia but not in acute lymphoblastic leukemia. A case control leukemia association study

Objective:Natural killers (NK) cell function is mainly controlled by the expression of killer immunoglobulin-like receptors (KIRs) and their ligation with the corresponding ligands. The objective of this study was to investigate the putative association of KIRs, HLA class I ligands, and KIR/ligand combinations with rates of development of acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), and chronic myeloid leukemia (CML).Materials and Methods:The KIR/HLA I genotypes of 82 patients with leukemia (ALL, n=52; AML, n=17; and CML, n=13) were determined by PCR-SSP method and compared with genotypes of healthy controls (n=126).Results:KIR genotype frequency differed significantly between myelogenous leukemia patients and healthy controls for KIR2DL5A (17.6% vs. 47.7%, p=0.02), KIR3DS1 (17.6% vs. 47.6%, p=0.02), and KIR2DS4*001 (36.6% vs. 20.2%, p=0.017). The incidence of homozygous HLA-BBw4 (31.0% vs. 12.5%, p=0.042) and HLA-Bw4Thr80 Thr80 (13.0% vs. 1.2%, p=0.01) was significantly elevated in myeloid leukemia patients compared to healthy controls. KIR/HLA class I ligand profile KIR3DS1(+)/L (-) was decreased and KIR3DL2(+)/HLA-A3/11(-) was increased among myeloid leukemia cases compared to controls.Conclusion:These data suggest that the activity of NK cells as determined by inherited KIR/HLA class I ligand polymorphisms influences the susceptibility to myelogenous leukemia, but not to lymphoblastic leukemia. Additionally, the KIR genotype characterized by the absence of the inhibitory KIR2DL2 and the activating KIR2DS2 and KIR2DS3 (ID2) was found at a lower frequency in patients compared to controls, which confirmed the need for complex analysis based on all possible KIR/HLA class I ligand polymorphism combinations.

Effect of secure clinical messaging on chemotherapy adherence.

e18542 Background: Oncologists offer essential clinical information to patients face-to-face or by phone. Today, patients commonly prefer digital communication. To test the effectiveness of this in patients, we deployed proprietary secure messaging (SM) to communicate clinical messages to patients regarding prescribed oral chemotherapy in an effort to improve adherence. Methods: Chronic myelogenous leukemia (CML) patients receiving a tyrosine kinase inhibitor (TKI) were evaluated as two groups: a test group of patients starting therapy between Jan - Jun 2018 who received SM within 45 days therapy start who received SM refill reminders, adherence, condition management, laboratory testing reminders every 30 days, and information regarding side effects, versus a control group that included propensity matched patients initiating therapy between Jan - June 2017 and who received only non-clinical text message refill reminders within 45 days of therapy start. Adherence was compared 6 months after the initial prescription. Primary outcomes were medication possession ratio (MPR) (total day supply/ [(last fill date+last fill day supply)-first fill date]), number of prescriptions filled, % of patients optimally adherent (MPR ≥ 85%), and rate of discontinuation after first fill. The secondary outcome was days of therapy. A t-test evaluated MPR and length of therapy; logistic regression compared discontinuation after first fill and % optimally adherent. Results: 478 CML patients opted into SM and 478 where in the control group. MPR improved 1.6% absolute points (93.20 control v 94.80 test, p = 0.04); no change was seen in number of prescriptions filled (5.04 control v 5.17 test, p = 0.19). More patients receiving SM were optimally adherent (83.05 control v 88.31 test, p = 0.03); discontinuation after first fill was unchanged (6.90 control v 6.49 test, p = 0.79), as were days of therapy (156.51 control v 159.22 test), p = 0.35). Conclusions: Patients initiated on TKI therapy with receiving clinical SM had significantly higher adherence than the control group and were more likely to be optimally adherent, despite the high adherence rates of CML patients at baseline. Further analysis is needed to understand the durability of this adherence improvement.

CLO19-029: Dosing Patterns of Nilotinib Use in SIMPLICITY, an Observational Study in Chronic Phase Chronic Myeloid Leukemia (CP-CML) Patients (Pts) in Routine Clinical Practice

Introduction: Dosing patterns of nilotinib (NIL) in chronic phase chronic myelogenous leukemia (CP-CML) patients (Pts) have not been well documented outside of clinical trials. SIMPLICITY (NCT01244750) is an ongoing observational study exploring tyrosine kinase inhibitor (TKI) use in routine clinical practice among CP-CML pts receiving TKIs in the US and Europe since 2010. This analysis reports NIL dosing patterns and explores predictors of dose reductions. A subset analysis focusing on the first-line (1L) approved dose of 300 mg twice daily (BID) will also be presented. Methods: Only SIMPLICITY pts receiving 1L NIL BID (n=349/408) were included. Baseline demographics and dosing patterns (starting dose, dose changes, time to dose reduction, and duration of therapy [DoT]) were analyzed descriptively. Statistical comparisons were made using t-tests, the Mann-Whitney U test for continuous variables, and chi-square for categorical variables. Logistic regression models were used to identify factors associated with dose reductions. Results: Of the 349 pts treated with 1L NIL, 281 (80.5%) started at the standard dose of 300 mg (BID) or the 400 mg (BID) dose for imatinib-resistance/intolerance, and 37 (10.6%) and 31 pts (8.9%) started on 150‒200 mg BID and 450‒800 mg BID. European pts were more likely to start on a dose >400 mg BID than US pts (P<.0001). Pts at academic centers were more likely to start on >400 mg BID than those treated at community practices (P<.0029). Among the pts starting NIL at 300 or 400 mg (BID) in 1L, 70.9% remained on these doses; 26.6% received a dose reduction (median time to dose reduction: 80.5 days); and 2.5% received a dose increase. Median DoT with NIL was 30.4 vs 43.9 months for pts with vs without a dose reduction (P=NS). The main reason for dose reduction was intolerance (n=51; 68.9%); in 51% of pts, a specific side effect was cited. Dose reductions were more likely in patients at academic centers (odds ratio=1.996; P=.021), but not in pts experiencing baseline fatigue (OR=1.799; P=0.072). Conclusions: Most pts treated with 1L NIL were started on 300 or 400 mg (BID); however, 1 in 4 pts required a dose reduction, most often due to intolerance. Physicians at academic centers were more likely to reduce the NIL dose than those in community practices. DoT with NIL for pts who received a dose reduction was shorter than that for those who did not. These findings will aid clinical decisions on dose optimization and maintaining response, whilst improving the patient quality of life.

Abstract 5860: Novel, first-in-class dual inhibitors of lysine specific demethylase 1 (LSD1) and histone deacetylatse 1 (HDAC) for treatment of cancer

Abstract Introduction: LSD1 is a flavin adenine dinucleotide (FAD)-dependent amine oxidase that removes methyl groups from mono- or dimethylated histone H3 lysine 4. Studies have shown that LSD1 contributes to acute myelogenous leukaemia pathogenesis and patients that are non responders to ATRA respond to LSD1-ATRA combination treatment in clinic. Recent studies have shown that there is cross-talk between two components of CoREST complex, LSD1 and HDAC1/2 and combined inhibition of these targets has been shown to be more efficacious in multiple cancers. Therefore, dual inhibitors targeting both LSD1 and HDAC could be useful in treating cancers. To test the hypothesis, we have developed a set of molecules that have dual activity on LSD1 and HDACs. Methods: To assess in vitro LSD1potency, TR-FRET assay was used. For assessing HDAC activity fluorescence based HDAC activity assay was performed. Western blotting and RT-qPCR were used to assess biomarkers of LSD1 and HDAC1/2 inhibition. Alamar blue cytotoxicity assay was used to assess cell proliferation. Xenograft models were used to assess in vivo efficacy. Results: Dual inhibitors show strong in vitro potency comparable to advanced clinical candidates, against LSD1. On HDAC1/2 these molecules showed comparable IC50 as marketed HDAC1/2 specific drugs. Interestingly, these dual inhibitors lead to stronger and complete inhibition of cell proliferation in broader types of cancer cell lines, including, acute myeloid leukemia, multiple myeloma, hepatocellular carinoma and small cell lung cancer when compared to LSD1 specific inhibitor GSK2879552. Target engagement studies confirm the concomitant increase in mRNA levels of GFI1b, CD11b, CD86 and H3K9 acetylation levels. In vitro activity also shows a clear translation in efficacy studies. JBI128, one of the lead molecules from this series was tested in multiple in vivo studies, including, non-small cell and small cell lung cancer, where it not only shows single agent activity, but can also be combined with other targeted agents or SoC safely. In addition, combination of JBI128 with immune checkpoint inhibitor anti-PDL1 antibody in the CT26 syngeneic model showed much stronger efficacy as compared to either of the single agents. This clearly suggests that these dual inhibitors in addition to being epigenetic modulators in the cancer cells also have a role in immune modulation thereby contributing to stronger efficacy. Further studies in terms of gene expression analysis are underway to understand the molecular mechanism behind the additional activity/efficacy of these dual inhibitors. Conclusion: These LSD1-HDAC dual inhibitors have the potential to be developed as first-in-class therapies that could offer stronger clinical efficacy compared to LSD1 or HDAC inhibitor alone. Further mechanistic and combination studies with standard therapies are in progress. Citation Format: Dhanalakshmi Sivanandhan, Sreekala Nair, Subramaniyam Tantry, Sridharan Rajagopal, Mahanandeesha Hallur, Kannan M, Srinatha KC, Damodara K, Dilipkumar M, Chandru G, Dimpy Ghosh, Pradeep N, Anuj Kumar Singh, Mohd Zainuddin, Rudresh G, Radha Sharma, Meghashree S, Durga P. Kumar, Purushottam Dewang, Sriram Rajagopal. Novel, first-in-class dual inhibitors of lysine specific demethylase 1 (LSD1) and histone deacetylatse 1 (HDAC) for treatment of cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5860.

Expression of CD47 in de novo acute myelogenous leukemia with normal karyotype and its clinical significance

Objective To investigate the CD47 expression in de novo acute myelogenous leukemia (AML) patients with normal karyotype and its clinical significance. Methods One hundred thirty-seven cases of de novo AML with normal karyotype and 3 healthy volunteers were selected. Relative CD47 expressions in normal bone marrow hematopoietic stem cells (HSC) and multipotent progenitor (MPP) from healthy volunteers, as well as bone marrow mononuclear cells (MNC) and leukemia stem cells (LSC, Lin- CD34+ CD38- CD90-) from AML patients were determined by flow cytometry. CD47 expression on the Lin- CD34+ CD38- LSC-enriched fraction of specimen was determined by flow cytometry. The FMS-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) was detected by using the Genome Analyzer platform. CD34+ CD38- CD47hi and CD34+ CD38- CD47lo expressing cells were identified and purified using FACS. Two groups of cells were inoculated with MethoCult H4445 medium on agarose-containing methylcellulose plates. After 12 days, MPP colony forming units (CFU) were counted, and 1×105 CD34+ CD38- CD47lo and CD34+ CD38- CD47hi cells were transplanted into NSG (NOD-SCID IL-2R γ null) mice irradiated by 280 cGy, and mice were sacrificed after 8 weeks. The ratio of human CD45+ cells was detected by flow cytometry. Results The expression of CD47 in AML patients was higher than that in the healthy control. CD47 was expressed in all FAB (French-American-British) subtypes of AML. No significant difference in CD47 expression among different FAB subtypes was found (F= 0.545, P > 0.05). Among the 37 patients with CD34+ CD38- CD47hi, 17 (46%) were FLT3-ITD negative, and 20 (54%) were FLT3-ITD positive. Among the 100 patients with CD34+ CD38- CD47hi, 63 (63%) cases were FLT3-ITD negative, 37 (37%) cases were FLT3-ITD positive. The rate of FLT3-ITD positive in patients with CD34+ CD38- CD47lo had no statistical difference compared with patients with CD34+ CD38- CD47hi (χ 2= 3.79, P > 3.79). The CD34+ CD38- CD47lo or CD34+ CD38- CD47hi which was selected by FACS, was inoculated with the methylcellulose plate containing agarose for 12 days, and CD34+ CD38- CD47lo cells could form CFU. The NSG mouse transplantation experiment showed that CD34+ CD38- CD47lo cells could be reconstructed hematopoiesis, and CD34+ CD38- CD47hi implantation failed. Conclusion CD34+ CD38- CD47hi could enrich LSC, which may be a potential marker to detect minimal residual disease. Key words: Leukemia, myelogenous, acute; Antigens, CD47; Colony-forming units assay

Correlation of patient prognosis with PU.1 and JDP2 to provide potential novel prognostic/diagnostic markers in MDS.

e18558 Background: PU.1 is a key transcription factor in haematopoiesis that plays important roles in various haematological malignancies. Previously, significant down-regulation of PU.1 has been reported in high risk myelodysplastic syndrome (MDS) and acute myelogenous leukemia (AML) patients. Methods: To clarify PU.1 molecular function we investigated the gene expression of PU.1 and JDP2 (c-Jun dimerization protein-2), a member of the activating protein-1 family located downstream of PU.1, in bone marrow from 12 MDS patients stratified according to IPSS-R score (6-low, 3-intermediate, 3-high risk), 1 AML patient and 10 normal controls. Samples were enriched for the mononuclear fraction by Ficoll separation. Total RNA was extracted and analysed by Real Time PCR for PU.1 and JDP2 expression relative to the housekeeping gene GAPDHusing the 2-ΔΔCT method. Results: We revealed both PU.1 and JDP2 are down regulated in MDS. In addition, our data suggests that PU.1 and JDP2 expression inversely correlates with disease, with expression of these genes consistently reducing according to IPSS-R groups. Furthermore, a positive correlation of PU.1 and JDP2expression < R = O.9333, s = 0.0004 > , provides additional evidence that suppression of JDP2 by PU.1 could contribute to the pathogenesis of AML. Notably, PU.1 and JDP2 do not correlate to the same extent in normal HSCs, indicating that cofactors are required for PU.1 to exert its JDP2-regulating function and that such cofactors are not present under normal conditions. To confirm that JDP2 suppression is a direct result of reduced PU.1 we performed PU.1-knockdown in K562 cells stably expressing PU.1 short interfering RNAs versus control cells. These analyses reveal only a partial reduction in JDP2 expression when analysed by RT-PCR and Western blot, suggesting a more complex regulatory mechanism. Additionally, both PU.1 and JDP2 expression was recovered by treatment with azacitidine, which is routinely used to treat MDS, suggesting an involvement in treatment response. Conclusions: PU.1 and JDP2 expression correlates with patients prognosis highlighting a potential role as new diagnostic and prognostic markers in MDS.

Treatment of acute lymphocytic leukemia in Down syndrome

AbstractDespite improvements in the prognosis of acute myelogenous leukemia in patients with Down syndrome, the prognosis of acute lymphocytic leukemia (DS-ALL) in these patients is still poorer than that of ALL in the general population. We compared treatment results between 4 patients with DS-ALL and 16 non-Down syndrome patients with ALL (NDS-ALL). Four patients with DS-ALL treated with TCCSG protocols achieved complete remission by the end of the induction phase. During the high-dose methotrexate (HD-MTX) phase, the dosage of MTX was reduced from 3 to 2 g/m2 after the syndrome of inappropriate ADH secretion developed in the first patient. All DS-ALL patients had severe mucositis and a longer period of anorexia than NDS-ALL patients, even after dose reductions, resulting in a longer HD-MTX phase in DS-ALL patients than in NDS-ALL patients. The mean dosages of 6-MP and MTX during the maintenance phase were significantly lower in DS-ALL patients than in NDS-ALL patients (6-MP 22.1 ± 9.9 mg/m2 and MTX 16....

Abstract IA01: The discovery of next-generation cancer targets and therapeutics

Abstract Cancer is driven by the accumulation of somatic genetic alterations that alter the activity of tumor suppressor and oncogenes. Together these activities lead to the dysregulation of cell proliferation, cell survival and metabolic pathways, among many others, that culminate in the acquisition of the transformed phenotype characteristic of malignant tumors. Reversing the consequences of oncogenic mutations through therapeutic intervention has been repeatedly demonstrated to bring significant patient benefit across a spectrum of oncogenes including BCR-ABL, BRAF, EGFR, cKIT, HER2, RET, ROS and ALK among others. Exemplary of both this paradigm is the longstanding experience of treating BCR-ABL+ chronic myelogenous leukemia (CML) patients with inhibitors of the Abelson kinase (ABL). Small clinical studies have recently shown that drug withdrawal can be associated with treatment free remission in a subset of patients achieving deep molecular responses. Towards the goal of eradicating CML, we have developed a novel potent and selective allosteric inhibitor of ABL, known as ABL001. ABL001 binds at a regulatory site typically occupied by a myristoyl group in wild-type ABL and inhibits ABL kinase activity through a mechanism distinct from catalytic site inhibitors. As a result, mutagenesis and genetic bar-coding experiments revealed a spectrum of resistance highly distinct from that seen with catalytic site inhibitors suggesting the possibility that co-administration of CML tumor cells with both types of inhibitors might act to block the development of therapeutic resistance. In vivo experiments showed that combination but not the single agent ABL001 and nilotinib could eradicate CML xenografts with sustained responses after treatment cessation. ABL001 is now in phase I clinical testing. Advances in next-generation sequencing and projects including the Cancer Genome Anatomy project and the International Cancer Genome Consortium have given us a detailed description of the genetic alterations across a broad spectrum of cancer. This however, does not immediately lead to a detailed functional understanding of those genes whose protein products are necessary for the maintenance of cancer viability. In order to more deeply understand the genes and pathways critical to the survival of cancer cells we have taken on the functional annotation of the Cancer Cell Line Encyclopedia (Nature 2012;483:603-7) through the use of deep pooled shRNA libraries. The resulting effort termed Project DRIVE (Deep RNAi-screening for Viablity Effects in cancer) has led to the generation of drop-out shRNA data for 7,500 genes targeted by 20 shRNAs per gene across 390 cancer cell lines. The data from this effort are highly robust identifying all known oncogenes mutated in the set of interrogated cell lines, along with multiple new cancer dependent features not previously described. Most recently, this project led to the discovery of PRMT5 as a novel dependency in cancers harboring co-deletion of MTAP and CDKN2A (Science 2016 351:1208-1) a finding that will be further described in the lecture. Citation Format: William Sellers. The discovery of next-generation cancer targets and therapeutics. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Targeting the Vulnerabilities of Cancer; May 16-19, 2016; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(1_Suppl):Abstract nr IA01.

Abstract 4442: Very low methylation is a novel epigenetic regulatory mechanism in normal tissues and predisposes to hypermethylation in cancer

Abstract High level DNA methylation of promoter CpG Island (CGI) represses gene expression. However, low-level CGI methylation is usually not distinguished from complete absence of methylation. Here we show that Very Low Methylated Regions (VLMRs 1-20%) on promoter CGIs are present in up to 20% of human and mouse genes and negatively correlate with gene expression in all tissues examined. In vitro, low-level methylation directly represses reporter gene expression; an effect mediated by methyl-CpG-binding proteins as transient knockdown of MBD4 reverses this repression. In vivo VLMR genes are enriched for polycomb occupancy but can also occur in H3K4me3 occupied promoters, where it correlates with gene repression independent of polycomb complex binding. VLMRs in promoter CGIs in healthy WBCs are 19 fold and 65 fold more likely to gain hypermethylation in Acute Myelogenous Leukemia and Myelodysplastic Syndrome patients respectively compared to unmethylated CGIs (<1%). Our study reveals a novel epigenetic control mechanism that has strong effects on gene expression in normal tissues and accounts for most of the gene specificity of hypermethylation in cancer. Citation Format: priyanka madireddi, Jaroslav Jelinek, Justin Lee, Takahiro Sato, Jean-Pierre Issa. Very low methylation is a novel epigenetic regulatory mechanism in normal tissues and predisposes to hypermethylation in cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4442.

Hematopoietic stem cell transplantation (HSCT) compared to consolidation chemotherapy (CT) to increase leukemia free survival (LFS) in acute myelogenous leukemia (AML) patients between 60 and 75 years irrespective of genetic risk: Report from the AML 2004 of the East German Study Group (OSHO).

e18501Background: HSCT has been reported to be a treatment option for elderly patients with AML. Here we analyze the outcome of CT compared to HSCT in elderly AML patients according to genetic risk...

Abstract 3509: Novel dual inhibitors of LSD1-HDAC for treatment of cancer

Abstract Introduction: LSD1 is a flavin adenine dinucleotide (FAD)-dependent amine oxidase that removes methyl groups from mono- or dimethylated histone H3 lysine 4. LSD1 has been reported to be overexpressed in many malignant tumors, including breast, colon, prostate, lung, gastric cancers and others. Studies have shown that LSD1 may contribute to acute myelogenous leukaemia pathogenesis and patients that are non responders to ATRA have responded to LSD1-ATRA combination treatment in clinic. Recent studies have shown that there is cross-talk between two components of CoREST complex, LSD1 and HDAC which provides an advantage to cancer cell proliferation and survival by regulating key signalling pathways. Accordingly, combined inhibition of LSD1 and HDAC has been shown to be more efficacious in inhibiting the growth of glioblastoma, AML and breast cancer. Therefore, dual inhibitors targeting both LSD1 and HDAC could be useful in treating several cancers effectively without enhancing systemic toxicity mediated by administration of multiple drugs. To test the hypothesis, we have developed a set of molecules that either have LSD1 activity alone or dual activity on LSD1 and HDAC. Methods: Computational chemistry approaches were used to design LSD1 specific and LSD1-HDAC dual inhibitors. To assess in vitro LSD1 potency, TR-FRET assay was used. For assessing in vitro HDAC activity fluorescence based pan-HDAC activity assay was performed. Western blotting was used to assess biomarkers of LSD1 and HDAC inhibition. Alamar blue cytotoxicity assay was used to assess cell proliferation. Results: In vitro potency of these compounds on LSD1 ranged between,0.007 to 0.2 μM, which was comparable to literature molecules. Pan-HDAC potency of these molecules in the in vitro assay was comparable to the approved drug, Vorinostat. H3K4 di- and mono-methyl markers and H3K9 acetylation levels respectively, were used as target engagement biomarkers for LSD1and HDAC inhibition. Treatment of leukaemia cells with LSD1or LSD1-HDAC inhibitors resulted in a dose-dependent increase in the levels of H3K4 di-and mono methylation. In addition, compounds with LSD1-HDAC dual activity showed a clear increase in H3K9 acetylation levels as well. In the cell proliferation assay, compounds with LSD1 activity alone did not show any effect up to 72 h of treatment. However, compounds with LSD1-HDAC dual activity showed a strong effect in inhibiting cell proliferation in multiple cell lines, with IC50s ranging from 0.02 to 2 μM, with leukaemia cell lines being generally more sensitive than solid tumors. Conclusion: The data obtained demonstrate that it is feasible to design dual LSD1-HDAC inhibitors that retain individual activity and potently inhibit cell proliferation, and such inhibitors could serve as powerful therapeutic agents for cancer. Further understanding of mechanisms to identify the right tool compound is in progress. Citation Format: Dhanalakshmi Sivanandhan, Sridharan Rajagopalan, Sreekala Nair, Purushottam Dewang, Durga Prasanna Kumar, Chandrika Mulakala, Lavanya Mahadevan, Neema Skariah, Venkata Giri Kavuru, Damodara Kuntrapaku, Suchitra Sajja, Mohammad Zainuddin, Krishnakumar V, Ritu Singh, Swarnakumari V, Ramachandraiah Gosu, Jayashree Aiyar, Pravin Iyer, Sriram Rajagopal. Novel dual inhibitors of LSD1-HDAC for treatment of cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3509. doi:10.1158/1538-7445.AM2015-3509

Abstract 1667: Targeting acute myelogenous leukemia with novel combrestatin analogs and development of predictors of response

Abstract Introduction: Most adult acute myelogenous leukemia (AML) patients relapse and die of the disease. AML stem cells (AML-SCs) have been postulated to resist standard chemotherapeutic agents and thus give rise to relapse. Combretastatin A-4 (CA-4), a natural product isolated from the South African tree Combretum caffrum, has been reported to possess anti-leukemic activity in AML cell lines. To increase the repertoire of combretastatin analogs with potent anti-leukemic activity, about 100 CA-4 analogs were tested in AML cells, and studied the mechanism that AML cells resist to such microtubule (MT) inhibitors. Methods: Cell viability/apoptosis was determined by annexin V and 7-AAD staining and flow cytometry. Cellular ROS production was measured by CellROX. Cell cycle was determined by DAPI staining and flow cytometry. Stem cell function was evaluated by colony assay and mouse xenograft assays. Tubulin depolymerization was measured by immunofluorescence or immunoblotting for microtubule fraction. Interaction of tubulin with BTAN or p38 MAPK was determined by pull down assays. Results: One of the most potent analogs, BTAN, was further investigated in a panel of leukemic cell lines and primary AML samples. Similar to CA-4, BTAN inhibited tubulin polymerization in vitro and induced MT depolymerization and cell cycle arrest at G2/M in AML cells. BTAN induced cell death of AML blast, progenitor and stem cells via caspase activation regardless of their proliferating status and level of intracellular reactive oxygen species (ROS). In addition, BTAN impaired the physical contact of AML with stromal cells. To further identify the mechanism of resistance to BTAN, we investigated several signal transduction pathways including AKT, JAK-STAT, ERK1/2, JNK and p38. p38 MAPK inhibitor sensitized leukemia cells to BTAN treatment, and the sensitivity of AML cells to BTAN correlated with the basal level of active/phosphorylated p38 MAPK, suggesting p38 MAPK may interfere with the sensitivity to BTAN in AML. We demonstrated that tubulin interacted with p38 MAPK, especially its phosphorylated form, which was disrupted by treatment with a p38 MAPK inhibitor. In addition, in the presence of p38 inhibitor, the accumulation of BTAN was potentiated in AML cells, suggesting activated p38 may interfere with the interaction between BTAN and its tubulin targets. Conclusion: Our data demonstrated that BTAN is a newly identified anti-AML-SC agent that works by disrupting the MT cytoskeleton and inducing caspase activation. These data suggest that the MT network is involved in the regulation of pro-survival signaling pathways even in quiescent AML-SCs, and that p38 MAPK is a key modulator for the response of AML blasts to microtubule inhibitors and that co-treatment with p38 MAPK inhibitors could be beneficial to microtubule destabilizing agents in AML. Note: This abstract was not presented at the meeting. Citation Format: Hongliang Zong, Narsimha R. Penthala, Vijayakumar N. Sonar, Paraskevi Giannakakou, Gail J. Roboz, Peter A. Crooks, Monica L. Guzman. Targeting acute myelogenous leukemia with novel combrestatin analogs and development of predictors of response. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1667. doi:10.1158/1538-7445.AM2015-1667

Integrated Genomic Profiling, Therapy Response And Survival in Adult Acute Myelogenous Leukemia

Purpose: Recurrent gene mutations, chromosomal translocations, and acquired genomic copy number aberrations (aCNA) have been variously associated with acute myelogenous leukemia (AML) patient outcome. However, knowledge of the co-occurrence of such lesions and the relative influence of different types of genomic alterations on clinical outcomes in AML is still evolving. Experimental Design: We performed SNP 6.0 array-based genomic profiling of aCNA/copy neutral loss-of-heterozygosity (cnLOH) along with sequence analysis of 13 commonly mutated genes on purified leukemic blast DNA from 156 prospectively enrolled non-FAB-M3 AML patients across the clinical spectrum of de novo , secondary, and therapy-related AML. Results: TP53 and RUNX1 mutations are strongly associated with the presence of SNP-A–based aCNA/cnLOH, while FLT3 and NPM1 mutations are strongly associated with the absence of aCNA/cnLOH. The presence of mutations in RUNX1 , ASXL1 , and TP53 , elevated SNP-A–based genomic complexity, and specific recurrent aCNAs predicted failure to achieve a complete response to induction chemotherapy. The presence of ≥1 aCNA/cnLOH and higher thresholds predicted for poor long-term survival irrespective of TP53 status, and the presence of ≥1 aCNA/cnLOH added negative prognostic information to knowledge of mutations in TET2 , IDH1 , NPM1 , DNMT3A , and RUNX1 . Results of multivariate analyses support a dominant role for TP53 mutations and a role for elevated genomic complexity as predictors of short survival in AML. Conclusions: Integrated genomic profiling of a clinically relevant adult AML cohort identified genomic aberrations most associated with SNP-A–based genomic complexity, resistance to intensive induction therapies, and shortened overall survival. Identifying SNP-A–based lesions adds prognostic value to the status of several recurrently mutated genes. Clin Cancer Res; 1–12. ©2015 AACR.

Modulation of lipid rafts (LR), p-glycoprotein (P-gp) and proinflammatory cytokines (PIC) with HMG-Co A reductase inhibitor simvastatin (SV) in chronic myelogenous leukemia (CML) patients with imatinib mesylate (IM) resistance.

e18032 Background: Primarily monocyte-derived cytokines and soluble interleukin-2-receptor (sIL2R) form of a group of PIC which are significantly elevated in patients with CML-cp or CML-bc. C-react...

Treatment and Management of Chronic Myelogenous Leukemia in Patients Cared for in a Large HMO

IntroductionCML management has rapidly changed in the last decade. Data are critically needed to characterize disease treatment, management, and drug use (particularly TKIs, tyrosine kinase inhibitors) to ultimately inform how to improve outcomes in patients with CML. Our objective was to describe practice trends in a large community based health plan serving over 3.2 million members in southern California, Kaiser Permanente (KPSC).MethodsPatients were identified from the health plan’s NCI-SEER affiliated cancer registry. The cohort consisted of 257 CML adult patients (18+ years, chronic phase, no prior cancer history) all diagnosed between January 2001 and December 2012 and followed through December 31, 2013. We collected data from electronic health records and chart reviews including demographics, TKI use and adherence, healthcare utilization and clinical outcomes. Medication possession ratio (MPR) was used to measure adherence, calculated as the number of days supplied divided by the number of days between first and last dispense date of any TKI. We classified patients into MPR>90% (indicates good adherence) and MPR<90%. Discontinuation was ascertained from providers’ notes in the medical charts.ResultsOf the 257 patients with newly diagnosed chronic phase CML, 111 were female (43%). About 58% (n=148) were non-Hispanic Whites, and 23% (n=59) were Hispanic. About half (48%, n=124) had one or more existing comorbidities. Over 90% of the cohort initiated TKI therapy within 3 months of diagnosis, and mean MPR was 88% (s.d. 18%). About two-thirds of the patients (63%, N=157) had an MPR>90% for TKI use. Virtually all patients (96%) started on imatinib. Of the 59 cases diagnosed 2010-2012, only 14 used dasatinib or nilotinib as first line therapy. Half of the cohort (n=107) discontinued first line treatment (mainly imatinib). Reasons for discontinuation included general adverse effects (e.g., skin rash, muscle cramps), cardiovascular, lung or abdominal effects (n=41); incomplete cytogenetic or molecular response (n=38); drug non-response, provider recommended, not compliant (n=21); or BMT/stem cell transplant (n=7).The cohort was followed a maximum of 13 years (mean 5.1 years, s.d. 3.1 years). The total number of hospitalizations was 351 among patients diagnosed 2001-2006; 113 diagnosed 2007-2009; and 71 diagnosed 2010-2012. The median length of each hospitalization was 10 days (s.d. 30.6) during the follow-up years. Among imatinib users, the median number of overall outpatient visits per year varied from 20 visits/year for patients diagnosed 2001-2006 to 11 visits/year for patients diagnosed 2010-2012. Overall, 18 patients underwent bone marrow and/or stem cell transplant (all diagnosed 2001-2006). We examined progression and overall survival in patients diagnosed in 2001-2006 and exposed to imatinib. The rate of progression to accelerated phase or blast crisis was lower in patients with MPR>90% (10.0/1,000 person-years) than with MPR<90% (14.2/1,000 person-years). Similarly, patients with MPR>90% had a lower mortality rate (25.9/1,000 person-years) than patients with MPR<90% (39.2/1,000 person-years). Kaplan Meier curves for overall survival also demonstrate better survival, but not significantly (log rank P=0.35), among those with MPR>90%. [Display omitted] DiscussionMost patients initiated TKI therapy within 90 days of diagnosis in this health plan. Our results show median length of each hospitalization was 10 days across all follow-up time. Despite having full pharmacy coverage, the percent of subjects with MPR>90% was moderate (64%). Our results also suggest that subjects with MPR>90% for TKIs had lower progression rates and greater overall survival, but the results were not significant, and need to be confirmed in a larger study. Next steps include examining the frequency of cytogenetic and molecular testing and how this might impact outcomes, and factors associated with lower TKI medication possession ratios. DisclosuresHaque:Novartis: Research Funding. Shi:Novartis: Research Funding. Chung:Novartis: Research Funding. Avila:Novartis: Research Funding. Chen:Novartis: stock options Other. Schottinger:Novartis: Research Funding.

Hyperdiploidy in AML: Outcomes of acute myelogenous leukemia (AML) patients (pts) with a hyperdiploid karyotype.

7088 Background: Hyperdiploidy with greater than 49 chromosomes is an infrequent phenomenon in AML, with a reported incidence of less than 2%. While autosomal loss is known to have poor prognosis, ...

DNA repair gene XRCC1 and XRCC3 polymorphisms and their association with chronic myelogenous leukemia

Objective: In the present study, because of its importance in DNA repair in cancer, we searched for polymorphisms of XRCC1 and XRCC3 genes in Turkish chronic myelogenous leukemia (CML) patients. Methods: In order to determine these gene polymorphisms, the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method was applied using genomic DNA extracted from samples. Results: The genotype frequencies were observed as follows: the XRCC1 Arg399Gln genotype frequencies were observed as 33.3% homozygote typical (G/G), 43.3% heterozygote (G/A) and 23.3 % homozygote atypical (A/A) in the 30 samples of patients group; the genotype frequencies of XRCC3 Thr241Met were found 50% homozygote typical (C/C), 36.7% heterozygote (C/T) and 13.3% homozygote atypical (T/T) in all samples. Conclusion: In this study, no statistical association was found between CML and XRCC1 Arg399Gln and XRCC3 Thr241Met polymorphisms in Turkish patients.