ALBERT

Description: The FDA-approved DNA hypomethylating agents (DHAs) like 5-azacytidine (5AC) and decitabine (DAC) demonstrate efficacy in the treatment of hematologic malignancies. Despite previous reports that showed histone acetylation changes upon using these agents, the exact mechanism underpinning these changes is unknown. In this study, we investigated the relative potency of the nucleoside analogs and non-nucleoside analogs DHAs on DNA methylation reversal using DNA pyrosequencing. Additionally, we screened their effect on the enzymatic activity of the histone deacetylase sirtuin family (SIRT1, SIRT2, SIRT3, SIRT5 and SIRT6) using both recombinant enzymes and nuclear lysates from leukemia cells. The nucleoside analogs (DAC, 5AC and zebularine) were the most potent DHAs and increased the enzymatic activity of SIRT6 without showing any significant increase in other sirtuin isoforms. ChIP-Seq analysis of bone marrow cells derived from six acute myeloid leukemia (AML) patients and treated with the nucleoside analog DAC induced genome-wide acetylation changes in H3K9, the physiological substrate for SIRT6. Data pooling from the six patients showed significant acetylation changes in 187 gene loci at different chromosomal regions including promoters, coding exons, introns and distal intergenic regions. Signaling pathway analysis showed that H3K9 acetylation changes are linked to AML-relevant signaling pathways like EGF/EGFR and Wnt/Hedgehog/Notch. To our knowledge, this is the first report to identify the nucleoside analogs DHAs as activators of SIRT6. Our findings provide a rationale against the combination of the nucleoside analogs DHAs with SIRT6 inhibitors or chemotherapeutic agents in AML due to the role of SIRT6 in maintaining genome integrity and DNA repair.

Description: Next generation sequencing (NGS) and single nucleotide polymorphism arrays (SNP-A) contribute to more precise identification of the spectrum of somatic mutations and karyotypic abnormalities in myeloid neoplasms. The diversity of individual defects and their combinations corresponds to the tremendous clinical heterogeneity. Identification of key driver genes remains a fundamental component to understanding the immense data generated from this technology and the contributions to leukemogenesis. In this project, we evaluated 1200 cases of MDS and AML. Somatic mutations of AT rich interactive domain 2 (ARID2) were found in myelodysplastic syndrome (MDS), myeloproliferative neoplasms (MPN), primary acute myeloid leukemia (pAML) or secondary AML (sAML). All ARID2 mutations occurred in either frameshift (at p.S1489 and p.T1645) or nonsense (at p.E65, p.S154 and p.Q1637) configurations, consistent with loss of function. We have identified a total of 5 mutant cases, all of somatic origin. Study of clonal architecture elucidated that ARID2 mutations were ancestral events in 50% of mutant cases as defined by variant allelic frequencies. By SNP-A, a commonly deleted region on chr.12q was defined by mapping minimally affected lesions in 9 patients with MDS, MPN, sAML or pAML. Haploinsufficient expression of ARID2 was confirmed by expression array analysis in the cases with del(12q), which is compatible to the frequent incidence of heterozygous ARID2 loss-of-function mutations. Del(12q) was more frequent in high-risk phenotype with higher blast counts. In addition, significantly lower expression of ARID2 was also observed in 22 out of 183 patients with diploid 12q. Interestingly, amplification of locus was not found in any of the cases studied by SNP-A. Altogether, such lesions of defective ARID2 were pathogenic in more than 10% of cases with myeloid neoplasms. ARID2 is encoding one of the components of SWI/SNF complex and involved in chromatin remodeling. Therefore, we stipulate that other genes which function together with ARID2 might be affected with somatic mutations or deletions. Further analyses demonstrated the presence of other somatic mutations and deletions affecting SWI/SNF complex, including ACTL6B (N=53) and SMARCD3 (N=66). While SWI/SNF complex lesions were mutually exclusive, concomitant subclonal mutations in such affected cases were commonly observed in RAS pathway genes, including K/NRAS, NF1 and PTPN11. To the contrary, ARID1B, which negatively regulates chromatin remodeling, is predominantly activated in the cohort with similar phenotype. While germline mutations of multiple genes in SWI/SNF complex are reported to be associated with Coffin-Siris syndrome, no family or past history characteristic of this congenital disorder was observed in our cohort. Further clues into the function of ARID2 in myeloid neoplasms came from studying splicing dysfunction in U2AF1 mutant cases. Deep RNA sequencing in the cases with U2AF1 mutations (p.S43F and p.Q157P), showed a consistent loss of ARID2 exon 8 (predominantly noted in sAML). Two types (whole and partial) of exon skipping led to frameshift in the transcript creating a stop codon. Targeted RT-PCR confirmed the transcriptome sequencing results in primary bone marrow samples of the cases with U2AF1 but not in the corresponding wild-type cases. These results are compatible with the genetic finding that spliceosomal mutations were not concomitantly observed in the cases with SWI/SNF complex defects, suggesting misspliced transcript with nonsense decay consequences is enough pathogenic to preclude additional spliceosomal mutations. To validate functional consequences of ARID2 loss, knockdown experiment using ARID2-shRNA transduction in K562 and HL60 cell lines were performed. Knockdown of ARID2 generally demonstrated cell cycle arrest in G2 phase prior to entry into the S-phase, which was partly caused by decreased expression of CDKL3 and CCND3. Hb staining with Benzidine showed impairment of erythroid differentiation in ARID2 knockdown K562, which was confirmed by cytological examination. In sum, multiple mechanisms of defective ARID2 including somatic mutations, haploinsufficiency and phenocopy due to spliceosomal mutations can be involved in ARID2-mediated leukemogenesis. Together with the other components, novel lesions of SWI/SNF complex constitute a group of tumor suppressor genes in myeloid neoplasms. Disclosures No relevant conflicts of interest to declare.

Description: Background: Minority patient (pt) populations are underrepresented in clinical trials and the proportion of such pts accrued to cancer studies has decreased, specifically among African Americans (AA) (Kwiatkowski et al. Cancer 2013). If minority pt populations have higher rates of comorbidities, restrictive eligibility criteria may contribute to systematic exclusion from studies. To explore the validity of this potential bias within AML pts, we characterized the comorbidity profile and compared outcomes between AA and white pts with AML. Methods: Adult (≥18 years) AML pts who received chemotherapy at Cleveland Clinic from 2003 to 2019 were included. The following characteristics were analyzed: age, sex, self-reported race, insurance, etiology of AML, comorbidities, hepatic/renal function tests, left ventricular ejection fraction (LVEF), and corrected QT interval (QTc). AML risk was categorized according to the 2017 European LeukemiaNet (ELN) risk stratification. Organ dysfunction was defined as ≥ grade 1 per the Common Terminology for Adverse Events. Fisher's exact and Welch's t-tests were performed to compare baseline characteristics. Multivariable logistic and Cox regression analyses were used to identify prognostic factors for response status per International Working Group criteria, and overall survival (OS). Kaplan-Meier method and log-rank test were used to estimate and evaluate OS, respectively. Results: Of 1,040 AML pts included, 939 (90.3%) identified as white and 101 (9.7%) as AA. Age, AML etiology, and AML risk were balanced between the two groups. Insurance coverage was different by race: AAs were more likely than whites to have Medicaid (11.9% vs. 5.1%), and less likely to have private insurance (16.8% vs. 34.6%), P1.5 x ULN vs. normal: HR= 1.69, P=.01). Clinically insignificant creatinine (〉ULN - 1.5 x ULN vs. normal: HR= 0.99, P=.97) and CrCl (84-60 ml/min vs. normal: HR=0.96, P=.69) abnormalities were not independently associated with OS. Subgroup analyses by race revealed similar results for AAs, although, there were no differences in OS based upon bilirubin. With the exception of liver comorbidities (OR= 0.17, P=.01), our analysis failed to identify significant evidence of association between response and comorbidities/organ dysfunction (similar results within the AA subgroup). Although AA were less likely to achieve a CR (AA vs. whites: OR=0.56, P=.05), there was no association between response and creatinine/CrCl abnormalities, regardless of severity. Conclusions: Within this cohort, renal function eligibility criteria may be an important barrier to enrollment, specifically within the AA population. Since there is no association between clinically insignificant renal laboratory values and OS or response, the liberalization of such criterion may be justified. Future trials that broaden the renal function eligibility criterion have the potential to accrue more diverse pt populations, which may reduce recruitment racial disparities and improve the generalizability of the trials' results. Disclosures Hobbs: Amgen: Research Funding; SimulStat Inc.: Consultancy. Mukherjee:Bristol-Myers Squibb: Speakers Bureau; Takeda: Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Projects in Knowledge: Honoraria; Celgene Corporation: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Partnership for Health Analytic Research, LLC (PHAR, LLC): Consultancy; McGraw Hill Hematology Oncology Board Review: Other: Editor. Advani:Glycomimetics: Consultancy, Research Funding; Kite Pharmaceuticals: Consultancy; Amgen: Research Funding; Pfizer: Honoraria, Research Funding; Macrogenics: Research Funding; Abbvie: Research Funding. Gerds:CTI Biopharma: Consultancy, Research Funding; Pfizer: Consultancy; Incyte: Consultancy, Research Funding; Roche: Research Funding; Celgene Corporation: Consultancy, Research Funding; Sierra Oncology: Research Funding; Imago Biosciences: Research Funding. Nazha:Daiichi Sankyo: Consultancy; Incyte: Speakers Bureau; Jazz Pharmacutical: Research Funding; Abbvie: Consultancy; Tolero, Karyopharma: Honoraria; Novartis: Speakers Bureau; MEI: Other: Data monitoring Committee. Sekeres:Celgene: Membership on an entity's Board of Directors or advisory committees; Syros: Membership on an entity's Board of Directors or advisory committees; Millenium: Membership on an entity's Board of Directors or advisory committees.

Description: SF3B1 mutations disrupt normal pre-mRNA splicing to cause disease. Drugs inhibiting the interaction between the SF3b complex and RNA or agents degrading auxiliary splicing factors are being tested as new avenues for targeted therapy in myeloid neoplasia (MN) with SF3B1 mutations. Here we describe the ability of small molecules to restore altered RNA processes in SF3B1MT MN. We previously reported (Visconte, ASH 2018) the identification of the small molecule 4-pyridyl-2-anilinothiazole (PAT) which showed growth inhibition of CRISPR/Cas9 SF3B1+/K700E cells and primary SF3B1MT cells. PAT did not influence the growth of other cell models without (THP1, MOLM13FLT3, OCIAML3DNMT3A, SIGM5TET2/DNMT3A, K562PHF6) and with other splicing factor mutations (K562U2AF1, K562LUC7L2). We now describe data from medicinal chemistry, transcriptome, and in vivo studies to advance drug development for SF3B1MT MN. SAR studies focused on logical and systematic modifications of PAT, e.g., i) replacement of the 2,4-disubstituted thiazole spacer ring with other heteroatom containing rings (5,6,7 membered aromatic or aliphatic ring structures); ii) alternative linking groups for the NH linker of the aniline of the tail region (sulfonamide, amide, substituted amine linkers); iii) alternative substituted aromatic and aliphatic ring structures for the phenyl head region substituent, led us to identify permissive sites for further chemical optimization. For example, a 4-chlorophenyl analog demonstrated activity [IC50, 3μM] similar to PAT. Competitive repopulation assays of bone marrow (BM) cells from dual reporters (ACTBtdTomato; EGFP) B6.GtROSA26 mixed with BM cells from conditional knock-in Sf3b1+/K700E mice injected in pre-lethally irradiated B6.SJL-PtprcaPepcb/BoyJ (CD45.1) recipients (n=18) were used as a preclinical murine model. This model then allowed i) demonstration of drug efficacy in reducing the competitiveness of SF3B1MT cells and ii) evaluation of therapeutic index in normal hematopoiesis. Post-transplant recovery, recipients of B6.GtROSA26 cells underwent PAT treatment (10 mg/Kg/IP/5 days weekly) for a period of 6 weeks without showing any signs of distress or drug intolerance (drop in blood count, weight loss, abdominal swelling, liver or kidney toxicity). Two weeks after transplantation, donor Sf3b1+/K700E cells had an engraftment capability similar to that of donor B6.GtROSA26 cells (83.6 ± 4 vs. 86.4 ± 2.4) when transplanted as a sole graft in CD45.1 recipients. PAT reduced almost half the percentage of Sf3b1+/K700E donor cells at 6 weeks of treatment (47.4%) vs. pre-treatment (83.6%). In mixed (1:1) BM transplants, Sf3b1+/K700E cellshad a repopulative disadvantage against competitors B6.GtROSA26 contributing for 16% of the marrow reconstitution. Similar to single graft transplants, PAT decreased the percentage of Sf3b1+/K700E cells at 6 weeks vs. pre-treatment (average, 6% vs. 16%) in chimeras. Consistent with the lack of toxicity of PAT treatment B6.GtROSA26 cells in chimeras were not affected by PAT and gradually repopulated the host (post-treatment, 80% vs. pre-treatment, 64%). Subsequently, we focused our efforts identifying important genes known to be dysregulated in MDS that were mostly influenced by drug treatment and minimally affected in normal cells. Our approach was based on the analyses of genes linked to erythropoiesis (a key hallmark of low-risk MDS). In normal hematopoiesis TGF-β signaling inhibits terminal erythroid maturation. Out of 13,775 genes, 5% (664/13,775) were found differentially expressed between CRISPR/Cas9 SF3B1+/K700E and parental cells of which 60% of these genes were significantly up-regulated and 40% down-regulated. Pathway analysis showed that the expression levels of SMAD family of genes and GDF factors changed significantly upon drug treatment. SMAD7 mRNA levels are 3-fold lower in MDS CD34+ cells (n=159) compared to the ones of healthy subjects (n=17) (GEO accession GSE58831) leading to TGF-β over activation. PAT treatment normalized SMAD7 expression levels in CRISPR/Cas9 SF3B1+/K700E cells by 3-fold while reducing the levels of GDF11. In summary, we have identified new drug entities that are modulators of transcriptomic changes which decrease the competitiveness of SF3B1MT cells. These results suggest combination therapies with current TGF-β pathway inhibitors. Disclosures Advani: Glycomimetics: Consultancy, Research Funding; Kite Pharmaceuticals: Consultancy; Amgen: Research Funding; Pfizer: Honoraria, Research Funding; Macrogenics: Research Funding; Abbvie: Research Funding. Kelly:Novartis, Bayer, Janssen, Pharmacyclics, Celgene, Astrazeneca, Seattle Genetics: Honoraria, Speakers Bureau; Takeda: Research Funding; Genentech, Verastem: Consultancy. Sekeres:Syros: Membership on an entity's Board of Directors or advisory committees; Millenium: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Maciejewski:Novartis: Consultancy; Alexion: Consultancy.

Description: Myelodysplastic syndromes (MDS) are unique among cancers because of the frequent occurrence of somatic mutations impacting spliceosome machinery. At least 65% of MDS patients harbor a mutation in one of several splicing factors including U2AF1, SF3B1 and SRSF2. Whole exome sequencing of MDS bone marrow uncovered somatic frameshift mutations in LUC7L2, the mammalian ortholog of a yeast splicing factor. LUC7L2 is located in the most commonly deleted region of chromosome 7. Deletions and frameshifts lead to haploinsufficient expression and therefore it can be approximated that a combined 14% of MDS patients have low expression of LUC7L2. Restoring expression of LUC7L2 in del(7q)-iPSCs partially rescues the differentiation of iPSCs into CD45+ myeloid progenitors. Although perhaps partly due to associated losses of other genes on chromosome 7, low expression of LUC7L2 correlates with a poorer patient prognosis, so its haploinsufficiency may play an important role in bone marrow failure. While U2AF1, SF3B1, and SRSF2 are well-characterized splicing factors, the function of LUC7L2 in pre-mRNA splicing is unexamined and its role in the MDS pathogenesis is undefined. We hypothesize that low expression of LUC7L2 results in the aberrant splicing of oncogenes and tumor suppressor gene transcripts thus reducing expression or altering function and contributing to the pathogenesis of MDS. We have characterized LUC7L2 as an alternative splicing regulatory protein that plays a repressive role in the regulation of alternative RNA splicing. We generated HEK-293 cells overexpressing V5-tagged LUC7L2 for immunoprecipitation-mass spectrometry, to ascertain protein interactions with LUC7L2. LUC7L2 co-immunoprecipitated with splicing regulators which are involved in splice site recognition. We performed cross-linking-IP-high-throughput-sequencing (CLIP-seq) to identify LUC7L2 binding sites on RNA. We identified 301 LUC7L2 RNA-binding sites as well as binding sites on U1 and U2 which is common for splicing regulatory proteins. Metagene analysis of these binding sites showed that LUC7L2 bound near splice sites in exonic sequences. We knocked down LUC7L2 expression in HEK293 and K562 cells to phenocopy the frameshifts and deletions observed in MDS patients. We used a PCR-based assay to measure the splicing efficiency of introns near LUC7L2-binding sites. Knockdown of LUC7L2 increased the splicing efficiency of 8/13 selected introns; this suggests that LUC7L2 represses selective splice site usage. We also performed RNA-seq to characterize global mis-splicing events. Analysis of RNA transcripts revealed a multitude of splicing changes, including enhanced exclusion of alternative introns. Knockdown LUC7L2 cells exhibited-altered expression of other splicing factors; this could have further contributed to the vast number of splicing changes observed. To identify specific splicing changes that could contribute to the pathogenesis of MDS, we compared the splicing profiles of LUC7L2-knockdown in K562 cells with RNA-seq data from K562 cells expressing U2AF1S34F, SRSF2P95H or SF3B1K700E. This analysis yielded several exon-skipping splicing patterns in cancer-relevant transcripts, such as oncogene PRC1, splicing factor PTBP1 and MRPL33. Additionally, we noticed commonly mis-spliced transcripts among the four datasets in which the missplicing events occurred in the functional domain, potentially conferring a functional change. Surprisingly, we observed missplicing of U2AF1 in LUC7L2-knockdown, SRSF2P95H, and SF3B1K700E K562 cells, which altered the length of the RNA-recognition UHM domain by inclusion of a mutually exclusive exon or retention of an intron. In this way, low expression of LUC7L2, or point mutants U2AF1S34F, SRSF2P95H, and SF3B1K700E,could alter U2AF1 function as a distal convergence point. In summary, we identified a novel splicing factor implicated in the pathogenesis of MDS. We characterized LUC7L2 as a splicing repressor and discovered many splicing changes caused by low expression of LUC7L2. Several genes were also mis-spliced in U2AF1S34F, SRSF2P95H and SF3B1K700E K562 cells targeting these for further study. Commonly mis-spliced targets such as U2AF1 may indicate that some of the novel therapeutics may have spliceosome mutation agnostic effects. If this applies to the LUC7L2 mutations, then they may also be effective in del7/del7q cases. Disclosures Carraway: Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; FibroGen: Consultancy; Jazz: Speakers Bureau; Novartis: Speakers Bureau; Amgen: Membership on an entity's Board of Directors or advisory committees; Balaxa: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Agios: Consultancy, Speakers Bureau. Sekeres:Opsona: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Opsona: Membership on an entity's Board of Directors or advisory committees. Saunthararajah:Novo Nordisk, A/S: Patents & Royalties; EpiDestiny, LLC: Patents & Royalties. Maciejewski:Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Apellis Pharmaceuticals: Consultancy; Ra Pharmaceuticals, Inc: Consultancy; Apellis Pharmaceuticals: Consultancy; Ra Pharmaceuticals, Inc: Consultancy.

Description: Lenalidomide (LEN) has established a new paradigm of targeted therapy in MDS. The mechanistic underpinnings of LEN efficacy are related to the synthetic lethality of this agent through its ability to bind cereblon (CRBN). LEN induces degradation of CK1α, which is encoded by the CSNK1A1 gene located on the del(5q) CDR, whereby haploinsufficient levels of this gene allow for selective toxicity to deletion mutants. Another common cytogenetic abnormality present in patients with myeloid neoplasia (MN) is -7/del(7q). To date no selective therapies exist for -7/del(7q), an urgent unfulfilled need, given the poor prognosis associated with this cytogenetic abnormality. We were interested to explore if this same notion of selective toxicity may be possible in del(7q) myeloid patients and sought to screen drugs for this focused population. From a large cohort of patients with MN (n=3,328), we found -7/del(7q) in 10% (n=316) of patients. We first identified a signature pattern of haploinsufficient genes on -7/del(7q) based on NGS. We then searched for haploinsufficient genes which, if targeted by investigational drugs, could provide a therapeutic window for selected MN subtypes in analogy to LEN in del(5q). For the purpose of our analysis, haploinsufficient expression was defined as

Description: Blastic Plasmacytoid Dendritic Cell Neoplasm (BPDCN) is an aggressive hematologic malignancy derived from plasmacytoid dendritic cells. The clinical presentation of BPDCN typically involves the skin at outset and invariably progresses to a leukemic phase with or without lymph node and splenic involvement. BPDCN blasts have a distinctive phenotypic appearance with ubiquitous overexpression of CD4, CD56, and CD123 (interleukin-3 receptor [IL-3R]). Although rare, BPDCN has been estimated to affect at least two thousand patients in the United States and Europe annually. There is no standard therapy for BPDCN, but treatment usually incorporates intensive combination chemotherapy, occasionally with allogeneic stem cell transplant. Treatment-naïve patients generally respond to these measures, but disease-free survival is brief, and most patients relapse with chemo-resistant disease. Despite aggressive upfront therapies, BPDCN has a dismal prognosis with estimated median survival of 9-14 months. SL-401, a novel biologic targeted therapy directed to IL-3R, is being developed to treat BPDCN, acute myeloid leukemia (AML), and several other IL-3R-expressing hematologic malignancies. SL-401, which is comprised of IL-3 conjugated to a truncated diphtheria toxin, a potent inhibitor of protein synthesis, has demonstrated ultra-high anti-tumor potency against BPDCN cell lines and primary BPDCN tumor cells, with IC50 values in the femtomolar (10-15 M) range and robust activity after treatment of an in vivo model of human primary BPDCN cell engraftment (Angelot-Delettre et al; ASH 2013). This report serves to update the results of SL-401 treatment in BPDCN patients who are participating in a Phase 1/2 study of SL-401 administered as a single cycle (15 minute infusion daily for 5 days). To date, 6 subjects with BPDCN (5 male/1 female; ages 35-72 years) received a single cycle of SL-401. The BPDCN blasts of all 6 patients expressed CD123 (IL-3R) as well as CD4 and CD56. Five patients had failed previous chemotherapy regimens, with 3 of these subjects also having received allogeneic stem cell transplantation, whereas one patient was treatment naïve. There have been no serious adverse events. Two patients had SL-401-related Grade 3 liver function test (LFT) elevations that recovered to Grade ≤2 within 24 hours and one patient had a brief episode of SL-401-related Grade 3 neutropenia and thrombocytopenia; all other SL-401-related adverse events (AEs) were Grade ≤2. One patient was not evaluable for response. To date, 5 (100%) of the 5 evaluable patients have had major responses. All five responding patients were treated with SL-401 at 12.5 µg/kg/day for 5 days, and experienced either a complete response (CR; 4 patients) or a partial response (1 patient). The CRs included disappearance of BPDCN in the skin, bone marrow, peripheral blood, spleen, and lymph nodes. As of August 2013, CR durations following a single cycle of SL-401 treatment are 11+ (ongoing), 5, 3, and 1 months; the PR duration is 1 month. Given these promising clinical responses to this targeted therapy, additional BPDCN patients are being accrued to this study and a pivotal program will begin in 2014. Disclosures: Frankel: Stemline Therapeutics: Research Funding. Woo:Angimmune: Patents & Royalties, Research Funding. Brooks:Stemline Therapeutics: Employment, Equity Ownership. Szarek:Stemline Therapeutics: Employment, Equity Ownership. Bergstein:Stemline Therapeutics: Employment, Equity Ownership, Patents & Royalties. Rowinsky:Stemline Therapeutics: Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees.

Description: Tipifarnib (T) exhibits modest activity in elderly adults with newly diagnosed acute myelogenous leukemia (AML). Based on preclinical synergy, a phase 1 trial of T plus etoposide (E) yielded 25% complete remission (CR). We selected 2 comparable dose levels for a randomized phase 2 trial in 84 adults (age range, 70-90 years; median, 76 years) who were not candidates for conventional chemotherapy. Arm A (T 600 mg twice a day × 14 days, E 100 mg days 1-3 and 8-10) and arm B (T 400 mg twice a day × 14 days, E 200 mg days 1-3 and 8-10) yielded similar CR, but arm B had greater toxicity. Total CR was 25%, day 30 death rate 7%. A 2-gene signature of high RASGRP1 and low aprataxin (APTX) expression previously predicted for T response. Assays using blasts from a subset of 40 patients treated with T plus E on this study showed that AMLs with a RASGRP1/APTX ratio of more than 5.2 had a 78% CR rate and negative predictive value 87%. This ratio did not correlate with outcome in 41 patients treated with conventional chemotherapies. The next T-based clinical trials will test the ability of the 2-gene signature to enrich for T responders prospectively. This study is registered at www.clinicaltrials.gov as #NCT00602771.

Description: Sequential administration of DNA methyltransferase (DNMT) inhibitors and histone deacetylase (HDAC) inhibitors has demonstrated clinical efficacy in patients with hematologic malignancies. However, the mechanism behind their clinical efficacy remains controversial. In this study, the methylation dynamics of 4 TSGs (p15INK4B, CDH-1, DAPK-1, and SOCS-1) were studied in sequential bone marrow samples from 30 patients with myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML) who completed a minimum of 4 cycles of therapy with 5-azacytidine and entinostat. Reversal of promoter methylation after therapy was observed in both clinical responders and nonresponders across all genes. There was no association between clinical response and either baseline methylation or methylation reversal in the bone marrow or purified CD34+ population, nor was there an association with change in gene expression. Transient global hypomethylation was observed in samples after treatment but was not associated with clinical response. Induction of histone H3/H4 acetylation and the DNA damage–associated variant histone γ-H2AX was observed in peripheral blood samples across all dose cohorts. In conclusion, methylation reversal of candidate TSGs during cycle 1 of therapy was not predictive of clinical response to combination “epigenetic” therapy. This trial is registered with http://www.clinicaltrials.gov under NCT00101179.