scholarly journals Crosstalk between the IGF1 and CXCL12 Pathways Defines Objective Responses to the Farnesyl Transferase Inhibitor Tipifarnib in AML and PTCL Patients

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 5349-5349
Author(s):  
Antonio Gualberto ◽  
Catherine Scholz ◽  
Vishnu Mishra ◽  
Matthew R Janes ◽  
Linda Kessler
Keyword(s):  
Gene Expression ◽  
Mechanism Of Action ◽  
Cancer Genomics ◽  
Lymphoid Cells ◽  
Rna Seq ◽  
Employment Equity ◽  
Cxcl12 Expression ◽  
Best Response ◽  
Equity Ownership ◽  
Pre Treatment

Abstract Background CXCL12 is a chemokine that is essential for the maturation of myeloid and lymphoid cells. Tipifarnib is a potent and selective inhibitor of the enzyme farnesyltransferase (FT). Treatment with this agent may translate to durable responses in subsets of patients (pts) with acute myeloid leukemia (AML), chronic myelomonocytic leukemia (CMML) and peripheral T-cell lymphoma (PTCL) but its mechanism of action in these indications is poorly understood. We have previously reported that tipifarnib interferes with the CXCL12 pathway. Here we show that an interplay between the CXCL12 and IGF1 pathways may define those pts who may experience objective responses with tipifarnib monotherapy. Methods Gene expression profile (GEP) data generated using RNA-Seq and the Affymetrix U133A gene-chips of tumor samples from 129 pts enrolled into tipifarnib trials (CTEP-20, KO-TIP-002,KO-TIP-004, INT-17) were analyzed with respect to study outcomes and complemented with analyses of mRNA expression in data sets from the cBioportal for Cancer Genomics. Gene expression was further validated using RT-PCR assays. RNA-Seq and Whole Exome Sequencing were conducted using standard methodologies. Clinical trial information: NCT00027872, NCT02464228. NCT02807272, NCT00354146. Results In order to improve our understanding of the molecular pathology of tumor CXCL12 overexpression, we investigated GEPs from 8,401 cancer pts in 25 studies available at cBioportal (TCGA, Provisional). Notably, we found a highly significant correlation in the expression of the IGF1 and CXCL12 genes in 19 of those studies. Intriguingly, the highest IGF1/CXCL12 correlations were observed in indications, including AML (ρ=0.698, p<0.001), in which activity of tipifarnib as monotherapy has been previously reported (AML, breast and urothelial cancer). Based on these results, we investigated the effect of IGF1/CXCL12 co-expression on pt outcome in tipifarnib studies. In previously untreated AML, 3 subsets of pts were identified with respect to bone marrow (BM) IGF1/CXCL12 expression: (1) high IGF1, high CXCL12 with predominantly hematological improvement or stable disease (SD) as best response, (2) intermediate IGF1, low CXCL12, with predominantly disease progression (PD), and (3) low IGF1, variable CXCL12, with 6 complete responses in 15 pts that were associated with CXCL12 expression (p=0.013), supporting the notion that CXCL12 pathway activation determines objective responses with tipifarnib while IGF1 mediates drug resistance. Barely detectable levels of IGF1 (and IGF2) were observed in the BMs of CMML pts in whom only 1 best response of PD was reported. In contrast, elevated levels of both CXCL12 and IGF1 were observed in PTCL pts responding to tipifarnib. Further investigation revealed that tumors of PTCL pts experiencing a partial response (PR) with tipifarnib expressed high levels of IGFBP7 (p=0.03), a natural inhibitor of the IGF1 receptor. Sequencing of the CXCL12 and IGF1 genes in PTCL samples revealed the presence of polymorphisms in non-responding pts: 8 pts, 7 carrying CXCL12 rs2839695 and 1 with a novel 3UTR variant, experienced a best response of PD. No pts with a best response of PR or SD carried 3UTR variants in CXCL12 (0% vs 80%, p=0.007). No pt with a best response of PR, 1 of 4 pts with SD and 6 of 10 pts with PD carried the IGFBP7 variant L11F (rs11573021) (16% PR/SD vs 60% PD, p=0.15) Conclusions Pre-treatment tumor CXCL12, IGF1 and IGFBP7 expression may enable the identification of pts susceptible to experience objective responses with tipifarnib monotherapy. These data may contribute to the understanding of the mechanism of action of FT inhibitors. Disclosures Gualberto: Kura Oncology: Employment, Equity Ownership. Mishra:Kura Oncology: Employment, Equity Ownership. Janes:Wellspring Biosciences: Employment, Equity Ownership. Kessler:Kura Oncology: Employment, Equity Ownership.

scholarly journals Chromatin Accessibility Mapping of Primary Erythroid Cell Populations Leads to Identification and Validation of Nuclear Factor I X (NFIX) As a Novel Fetal Hemoglobin (HbF) Repressor

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 812-812
Author(s):  
Mudit Chaand ◽  
Chris Fiore ◽  
Brian T Johnston ◽  
Diane H Moon ◽  
John P Carulli ◽  
...  
Keyword(s):  
Gene Expression ◽  
Globin Gene ◽  
Chromatin Accessibility ◽  
Cell Populations ◽  
Rna Seq ◽  
Globin Mrna ◽  
Employment Equity ◽  
Equity Ownership ◽  
Erythroid Maturation ◽  
Globin Gene Expression

Human beta-like globin gene expression is developmentally regulated. Erythroblasts (EBs) derived from fetal tissues, such as umbilical cord blood (CB), primarily express gamma globin mRNA (HBG) and HbF, while EBs derived from adult tissues, such as bone marrow (BM), predominantly express beta globin mRNA (HBB) and adult hemoglobin. Human genetics has validated de-repression of HBG in adult EBs as a powerful therapeutic paradigm in diseases involving defective HBB, such as sickle cell anemia. To identify novel factors involved in the switch from HBG to HBB expression, and to better understand the global regulatory networks driving the fetal and adult cell states, we performed transcriptome profiling (RNA-seq) and chromatin accessibility profiling (ATAC-seq) on sorted EB cell populations from CB or BM. This approach improves upon previous studies that used unsorted cells (Huang J, Dev Cell 2016) or that did not measure chromatin accessibility (Yan H, Am J Hematol 2018). CD34+ cells from CB and BM were differentiated using a 3-phase in vitro culture system (Giarratana M, Blood 2011). Fluorescence-activated cell sorting and the cell surface markers CD36 and GYPA were used to isolate 7 discrete populations, with each sorting gate representing increasingly mature, stage-matched EBs from CB or BM (Fig 1A, B). RNA-seq analysis revealed expected expression patterns of the beta-like globins, with total levels increasing during erythroid maturation and primarily composed of HBB or HBG transcripts in BM or CB, respectively (Fig 1C). Erythroid maturation led to progressive increases in chromatin accessibility at the HBB promoter in BM populations. In CB-derived cells, erythroid maturation led to progressive increases in chromatin accessibility at the HBG promoters through the CD36+GYPA+ stage (Pops 1-5). Chromatin accessibility shifted from the HBG promoters to the HBB promoter during the final stages of differentiation (Pops 6-7), suggesting that HBG gene activation is transient in CB EBs (Fig 1D). Hierarchical clustering and principal component analysis of ATAC-seq data revealed that cell populations cluster based on differentiation stage rather than by BM or CB lineage, suggesting most molecular changes are stage-specific, not lineage-specific (Fig 2A, B). To identify transcription factors driving cell state, and potentially beta-like globin expression preference, we searched for DNA binding motifs within regions of differential chromatin accessibility and found NFI factor motifs enriched under peaks that were larger in BM relative to CB (Fig 2C). Transcription factor footprinting analysis showed that both flanking accessibility and footprint depth at NFI motifs were also increased in BM relative to CB (Fig 2D). Increased chromatin accessibility was observed at the NFIX promoter in BM relative to CB populations, and in HUDEP-2 relative to HUDEP-1 cell lines (Fig 2E). Furthermore, accessibility at the NFIX promoter correlated with elevated NFIX mRNA in BM and HUDEP-2 relative to CB and HUDEP-1, respectively. Together these data implicated NFIX in HbF repression, a finding consistent with previous genome-wide association and DNA methylation studies that suggested a possible role for NFIX in regulating beta-like globin gene expression (Fabrice D, Nat Genet 2016; Lessard S, Genome Med 2015). To directly test the hypothesis that NFIX represses HbF, short hairpin RNAs were used to knockdown (KD) NFIX in primary erythroblasts derived from human CD34+ BM cells (Fig 3A). NFIX KD led to a time-dependent induction of HBG mRNA, HbF, and F-cells comparable to KD of the known HbF repressor BCL11A (Fig 3B-D). A similar effect on HbF was observed in HUDEP-2 cells following NFIX KD (Fig 3E). Consistent with HbF induction, NFIX KD also increased chromatin accessibility and decreased DNA methylation at the HBG promoters in primary EBs (Fig 3F, G). NFIX KD led to a delay in erythroid differentiation as measured by CD36 and GYPA expression (Fig 3H). Despite this delay, by day 14 a high proportion of fully enucleated erythroblasts was observed, suggesting NFIX KD cells are capable of terminal differentiation (Fig 3H). Collectively, these data have enabled identification and validation of NFIX as a novel repressor of HbF, a finding that enhances the understanding of beta-like globin gene regulation and has potential implications in the development of therapeutics for sickle cell disease. Disclosures Chaand: Syros Pharmaceuticals: Employment, Equity Ownership. Fiore:Syros Pharmaceuticals: Employment, Equity Ownership. Johnston:Syros Pharmaceuticals: Employment, Equity Ownership. Moon:Syros Pharmaceuticals: Employment, Equity Ownership. Carulli:Syros Pharmaceuticals: Employment, Equity Ownership. Shearstone:Syros Pharmaceuticals: Employment, Equity Ownership.

Roar: A Phase Ib Trial of Oral Azacitidine (CC-486) in Combination with Lenalidomide and Dexamethasone (Rd) for Patients with Relapsed and/or Refractory (R/R) Multiple Myeloma Who Have Failed a Prior Lenalidomide-Containing Regimen

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2121-2121
Author(s):  
Anna Kalff ◽  
Kate Reed ◽  
Tiffany T. Khong ◽  
Sridurga Mithraprabhu ◽  
Malarmathy Ramachandran ◽  
...  
Keyword(s):  
Median Time ◽  
Dna Methyltransferase ◽  
Progression Free Survival ◽  
Maximum Tolerated Dose ◽  
Employment Equity ◽  
Dose Escalation Study ◽  
Phase Ib ◽  
Best Response ◽  
Equity Ownership ◽  
Pre Treatment

Abstract Introduction Azacitidine (AZA) is a DNA methyltransferase inhibitor and cytotoxic agent with survival benefits in higher-risk MDS and AML. We have demonstrated that AZA rapidly induces apoptosis of MM cells, and induces synergistic killing when combined with lenalidomide (LEN). This, coupled with development of an oral AZA formulation (CC-486), provides rationale for pursuing this combination in MM. The cereblon [CBRN]-Ikaros/Aiolos-IRF4/c-MYC signalling pathway has been implicated in the mechanism of action of cytotoxicity for IMiD®compounds. This pathway, as well as immune markers (CD8, PD-1) may represent potential biomarkers for predicting outcomes/response to IMiD® compounds. Aims: In R/R myeloma patients who previously failed a LEN-containing regimen: to determine the maximum tolerated dose (MTD) of CC-486 in combination with Rd; to characterise safety/tolerability; to assess efficacy: overall response rate (ORR), progression free survival (PFS), overall survival (OS). To correlate pre-treatment MM protein expression (CRBN, Ikaros, Aiolos, IRF4, c-MYC), as well as whole marrow expression of CD8/PD-1 with response and survival (PFS/OS) in patients receiving CC-489 plus Rd. Methods: Phase Ib, single centre, 3 x 3 dose escalation study. LEN 25mg d1-21 and dexamethasone 40mg d1, 8, 15, 22 of 28 day cycle were combined with escalating doses of CC-489: initial dose 100mg for d1-14, increasing by either 7 days or 50mg/cohort, to a maximum of 200mg d1-21. Dose limiting toxicity (DLT) was assessed during cycle 1. Treatment continued until toxicity/progression. Single- and dual-IHC assays were performed on pre-treatment trephines (Single: CD8, PD-1; Dual: CD138 paired with CRBN, Aiolos, Ikaros, IRF4, c-MYC). In samples with >10% CD138, presence and sub-cellular localisation of each stain was documented, and given an H score: product of % of tumour positive and intensity of staining (0-3). Single CD8 and PD-1 IHC assays were reported as positive cells/mm2. Samples were grouped according to quartiles (Group1: lower quartile, Group2: mid-range, Group3: upper quartile) and analysis was performed using SAS statistical platform v9.4. Results: 22 patients commenced therapy (F=10, M=12), median age 67yrs (50-82yrs). Median prior lines of therapy: 5 (2-8), including 16 ASCT and 2 prior allogeneic transplant. All had failed LEN (R/R=18, primary refractory (PrimR)=4), 10/22 received and failed pomalidomide (POM) (R/R=4, PrimR=6). All had received bortezomib (R/R=10, PrimR=10), 18/22 were both bortezomib and LEN refractory. CC-486 dose reached was 200mg d1-21, with no DLTs observed at time of reporting. One patient died due to unrelated causes prior to end cycle 1, therefore was not evaluable for response. ORR (≥PR) was 43% (9/21): 8 PR, 1 VGPR. Of the remaining patients, 2 achieved MR, 5 SD, and 5 PD. (clinical benefit rate (≥MR) = 52%). Median time to best response in patients with ≥MR: 2.5m (1-3.7m). Responses were seen in cohorts: [100mg d1-14 (3PR=3), 100mg d1-21 (VGPR=1, PR=3), 150mg d1-21 (PR=2, MR=2)]. Median time on study: 3m; responders (≥MR): 6.3m (2.5-15m), non-responders: 1.7m (0.9-8m). Median PFS 3m, median OS 15m. One patient remains on study. 3/6 patients treated with LEN in prior 1-2 treatment lines responded (PR=2, VGPR=1), 1/6 had SD. 1/10 patients treated with POM in prior 1-2 treatment lines responded (PR), with 2 achieving MR and 3 SD. Patients with a lower expression of cMYC and IRF4 had superior PFS compared with patients with higher expression (cMYC: Group1 vs Group3 p=0.052; IRF4: Group1 vs Group2 p=0.04). Patients with lower numbers of CD8+ T-cells had better PFS than those with higher (p=0.069). There was no association between degree of expression of CBRN, Ikaros, Aiolos or PD-1 and survival (PFS/OS). As CRBN expression increased, patients were more likely to respond (≥PR) (p=0.07) and patients with low PD-1 expression were more likely to respond (≥ MR) (Group1 vs Group3 p=0.05). Conclusion CC-489 combined with Rd is well tolerated and effective with durable responses in a subset of heavily pre-treated R/R MM patients, including those who recently failed IMiD® compound therapy, suggesting that CC-489 may overcome drug resistance. IHC may have utility in identifying subsets of patients more likely to respond to CC-489 and Rd (CRBN, PD-1) and predict survival (cMYC, IRF4 and CD8). This trial has been expanded to include other sites and less heavily pre-treated patients. Disclosures Thakurta: Celgene: Employment, Equity Ownership. Wang:Celgene: Employment. Guzman:Celgene: Employment. Cuoto:Celgene: Employment. Ren:Celgene: Employment, Equity Ownership.

scholarly journals Baseline Tumor Transcriptome Characteristics Associated with the Response of Relapsed/Refractory Hodgkin Lymphoma Patients to Brentuximab Vedotin in Combination with Nivolumab

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2837-2837
Author(s):  
Daniel E. Zak ◽  
Carol Anne Anne Ogden ◽  
Alex F. Herrera ◽  
Mariana Sacchi ◽  
Matt Onsum
Keyword(s):  
Gene Expression ◽  
Tumor Microenvironment ◽  
Research Funding ◽  
Brentuximab Vedotin ◽  
Rna Seq ◽  
Employment Equity ◽  
Expression Ratio ◽  
Equity Ownership ◽  
Tumor Expression ◽  
Tumor Biopsies

Abstract Introduction: In recently-reported interim results from a phase 1/2, open-label, multicenter study (NCT02572167), we showed that 61% of relapsed/refractory Hodgkin Lymphoma (HL) patients achieved complete responses (CRs) after treatment with Brentuximab Vedotin (BV) in combination with Nivolumab (Nivo) (Herrera et al., 2018). To gain insight into tumor microenvironment driven disease characteristics associated with CRs after combined BV+Nivo therapy, we performed RNA sequencing (RNA-Seq) transcriptome analysis of formalin-fixed paraffin-embedded tumor biopsies obtained from 37 study participants (23 CR, 14 PR/SD/PD) prior to the start of treatment. Results: Within a set of 132 candidate genes comprised of BV+Nivo therapeutic targets (CD30, PD-1, PD-L1), the markers for immune cells, inflammatory response factors, and potential resistance mechanism factors within the tumor microenvironment, the gene most strongly associated with CRs was the BV target CD30 (TNFRSF8) (p=4x10-4, FDR=5%). Baseline tumor expression of CD30 above 16 counts per million mapped reads (CPM) was strongly associated with achieving CR after BV+Nivo (65% sensitivity and 93% specificity). CRs were associated with several other factors, including higher baseline tumor expression of macrophage markers CD14 and CD163 and lower expression of the Nivo target PD-1 (PDCD1) and multidrug resistance-associated protein 2 (MRP2/ABCC2) in the tumor microenvironment. Both PD-1 and MRP2 were significantly complementary to CD30 for discriminating CRs from the other patients on the basis of baseline gene expression profiling in tumor microenvironment (p<0.01, FDR<20%), and a simple baseline gene expression ratio of CD30/PD-1 achieved high accuracy (area under the receiver operating characteristic curve = 0.93). Interestingly, baseline expression of PD-L1 (CD274) was not significantly associated with development of CRs after BV+Nivo, either by itself (p=0.13) or in combination with CD30 (p=0.22), which may be expected given that consistently high PD-L1 expression has been reported for HL. To gain further insight into the role of the tumor microenvironment (TME) in determining HL responsiveness to BV+Nivo, we performed mathematical deconvolution of the baseline HL biopsy transcriptomes. This analysis suggested that both macrophages and T cells were major leukocyte constituents of the HL biopsy TMEs, with higher baseline macrophage abundance and lower baseline T cell abundance being associated with higher CR rates after BV+Nivo treatment. Conclusions: The CR rate for combination therapy of BV+Nivo in relapsed/refractory HL is nearly double the rate reported for this patient population for either agent individually, suggesting the possibility of complementary mechanisms between BV-targeted and Nivo-targeted pathways. By performing RNA-Seq analysis of patient baseline tumor biopsies from the BV+Nivo trial (NCT02572167), we were able to identify specific tumor characteristics that are associated with favorable responses to treatment. In particular, the gene expression ratio of CD30/PD-1 was an accurate discriminator for patients that would achieve CRs. This result suggests that BV+Nivo efficacy may be observed in tumors in which the potential for BV-driven immunogenic cell death (ICD) - as indicated by CD30 expression - exceeds a threshold set by the immunosuppressive potential of the tumor (as indicated by PD-1 expression). This hypothesis is supported by the deconvolution analysis results that implicated a favorable role for macrophages (which may become activated in response to BV-induced ICD) in the tumor and a detrimental role for immunosuppressive tumor T cells. Immunohistochemical analyses of biopsies from the study are underway to quantify target abundance and to further characterize the TMEs. These data will be integrated with the transcriptomics to validate and refine the model for BV+Nivo efficacy in relapsed/refractory HL. Disclosures Zak: Seattle Genetics: Employment, Equity Ownership. Ogden:Seattle Genetics: Employment, Equity Ownership. Herrera:Pharmacyclics: Consultancy, Research Funding; Seattle Genetics: Research Funding; Immune Design: Research Funding; Bristol-Myers Squibb: Consultancy, Research Funding; Merck, Inc.: Consultancy, Research Funding; AstraZeneca: Research Funding; Genentech: Consultancy, Research Funding; KiTE Pharma: Consultancy, Research Funding; Gilead Sciences: Research Funding. Sacchi:Bristol-Myers Squibb: Employment, Equity Ownership. Onsum:Seattle Genetics: Employment, Equity Ownership.

scholarly journals Cell-of-Origin Subtype Prediction of Diffuse Large B-Cell Lymphoma Using Gene Expression and Proteomic Data

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1712-1712
Author(s):  
Huiqing Liu ◽  
Jin Lu ◽  
Zhiwan Dong ◽  
Hao Liu ◽  
Edward Salinas ◽  
...  
Keyword(s):  
Gene Expression ◽  
B Cell ◽  
Expression Profiles ◽  
B Cell Lymphoma ◽  
Training Data ◽  
Rna Seq ◽  
Training Set ◽  
Employment Equity ◽  
Equity Ownership ◽  
Research Institute

Abstract Introduction Diffuse large B-cell lymphoma (DLBCL) subtypes can be identified based on immunohistochemistry, somatic mutation and gene expression profiles. These cell-of-origin (COO) subtypes have distinct biological and pathogenic characteristics. In addition, studies have shown the association of COO with drug response such as with rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) as well as targeted therapy. Therefore, proper assessment of COO subgroup is an important step in treatment selection and outcome. In this study, we sought to develop predictive COO models using RNA-Seq based gene expression profiling and plasma proteomic data, focusing on the two defined major DLBCL subtypes - germinal center B cell-like (GCB) and activated B cell-like (ABC). Methods COO subgroups of patient samples were assigned by the Hans algorithm. Data from archival formalin-fixed paraffin-embedded (FFPE) tissues were obtained using the Illumina HiSeq platform (RNA-Seq). A subset of samples were used as a training set to select differentially expressed genes (DEGs) in ABC vs. GCB lymphomas to build support vector machine (SVM) classification models. The model with best leave-one-out cross validation (LOOCV) on the training set was applied to the remaining samples to assess its initial predictive power. Gene set enrichment analysis (GSEA, Broad Institute) and key pathway analysis (KPA, Clarivate Analytics) were also utilized to further explore the underlying biology of each COO subtype. Protein expression data using the Olink Proteomics platform was obtained from baseline patient plasma samples. Protein biomarkers to differentiate ABC and GCB subgroups were identified from a set of training samples and evaluated in independent cohorts. Due to notable batch effect, batch information was included and specified as a random factor in the model. Results Genes identified by Scott et al. (Blood 2014) for COO assignment were first tested in our RNA-Seq training data of 6 GCB and 8 ABC samples. Thirteen of 15 gene markers showed significant differences between the ABC and GCB subgroups. From these markers, we further selected 6 to build machine learning models based on fold change, false discovery rate and entropy. This 6-gene signature include 3 markers relatively up-regulated in ABC subtype and 3 up-regulated in GCB subtype. A SVM model with these genes achieved 100% LOOCV on the training data and correctly predicted COO of 20/22 samples in the validating cohort with 1 GCB and 1 ABC samples misclassified. These two samples were also misclassified if a larger panel of signature genes from Scott et al. (Blood 2014) was used. KPA on the DEGs from ABC vs. GCB predicted the activation of NFKB1and STAT4/5 transcription factors as key elements upstream of the DEGs, indicating promoted signaling of NFкB and STAT pathways in ABC subgroup. On the other hand, REST was predicted as an inhibited upstream regulator of some DEGs. RCOR1, a corepressor of REST, has significantly lower expression level in the ABC subgroup in our data. These may imply the inhibition of REST/RCOR1 pathway in ABC patients. Plasma protein data from two studies were used to form a training set with 21 GCB and 6 ABC. A set of differentially expressed analytes from ABC vs. GCB were identified which included several targets of the NFкB pathway. In an independent cohort containing 5 GCB and 4 ABC plasma samples, many of these same plasma proteins showed differential expression profiles between ABC and GCB, making them potential blood-based biomarkers for COO determination. Conclusions In this study, we built a SVM model with a subset of genes from Scott et al. (Blood 2014) to accurately predict COO of refractory DLBCL from archival FFPE tissue. Further analyses of the RNA-Seq data disclosed alterations in key transcriptional hubs between the different COO subgroups. Olink plasma data from independent cohorts demonstrated potential protein markers for a plasma-based differentiation of the ABC and GCB subtypes. These biomarkers and machine learning models are being further validated using additional datasets. Disclosures Liu: Incyte Research Institute: Employment, Equity Ownership. Lu:Incyte Research Institute: Employment, Equity Ownership. Dong:Incyte Research Institute: Employment, Equity Ownership. Liu:Incyte Research Institute: Employment, Equity Ownership. Salinas:Incyte Research Institute: Employment, Equity Ownership. Owens:Incyte Research Institute: Employment, Equity Ownership. Pratta:Incyte Research Institute: Employment, Equity Ownership. Smith:Incyte Research Institute: Employment, Equity Ownership. Tada:Incyte Research Institute: Employment, Equity Ownership. Newton:Incyte Research Institute: Employment, Equity Ownership. Burn:Incyte Research Institute: Employment, Equity Ownership.

scholarly journals Hotspot Mutations in SF3B1 Result in Increased Alternative Splicing in Multiple Myeloma and Activation of Key Cellular Pathways

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4454-4454
Author(s):  
Michael A Bauer ◽  
Cody Ashby ◽  
Christopher Wardell ◽  
Maria Ortiz ◽  
Erin Flynt ◽  
...  
Keyword(s):  
Gene Expression ◽  
Alternative Splicing ◽  
P Value ◽  
Rna Seq ◽  
Splice Sites ◽  
Differential Splicing ◽  
Employment Equity ◽  
Differential Gene ◽  
Equity Ownership ◽  
Hotspot Mutations

Abstract Introduction: Mutations in the components of the spliceosome have been shown to occur at relatively high frequency in many cancers such as chronic lymphocytic leukemia, myelodysplastic syndromes and breast cancer. One component in particular, encoded by SF3B1, has hotspot missense mutations that result in a significant increase in alternatively spliced transcripts. RNA splicing in Multiple Myeloma (MM) has not been investigated and in particular the extent of mutations in SF3B1 and its effects on the transcriptome. Methods: Using the MMRF CoMMpass dataset (N=1273) of newly diagnosed MM patients, samples with whole exome sequencing (WES) were analyzed for mutations using Strelka and Mutect, and samples with SF3B1 mutations identified. A range of approaches were used to explore the effect of the SF3B1 mutations on the transcriptome and to determine possible downstream effects. Using RNA-seq with matched WES samples (n=615), the splice junction usage of SF3B1 mutants was compared against non-mutated samples which were matched for key MM molecular sub-types. The RNA-seq data was analyzed using a pipeline that included STAR and Salmon, aligning to human reference genome hg38, gene and transcript differential expression analysis tools DESeq2 and StringTie/Ballgown, differential splicing exon usage tools JunctionSeq/QoRTs, DEXSeq, and SUPPA and for Gene Set Enrichment Analysis (GSEA) the R package FGSEA was used. Results: From the WES data 1.7% (22/1273) of samples had mutations in SF3B1 of which 5 had mutations in the hotpot codons of K666 and K700. Differential isoform analysis of the 22 SF3B1 mutant samples compared to non-mutated samples did not identify any transcripts. However, when the analysis was restricted to the 5 samples with hotspot mutations differential gene expression identified 146 genes that were significantly differentially expressed at an adjusted p-value <0.05. Additionally, many genes that did not show an overall gene expression change between the control and the SF3B1 hotspot mutants did at transcriptional level where we observed isoform switching which included the protein coding genes BCL2L1, SNUR, ACKR3 and CRLF2. Results of differential gene analysis between the control and SF3B1 mutants were used in GSEA and significant normalized enrichment scores (NES) identifying increased protein secretion (p-value =0.009, NES= 1.9) and unfolded protein response (UPR) (p-value = 0.02, NES = 1.52) pathways. Conversely GSEA identified decreased apoptosis (p-value = 0.008, NES = -1.76), KRAS signaling (p-value = 0.008, NES = -1.92), TNFA signaling via NF-κB (p-value = 0.008, NES= 2.12) pathways in SF3B1 mutant samples. Investigation of splicing loci revealed that novel splice loci were significantly more abundant in the SF3B1 mutants versus control samples. Differential splicing analysis detected 474 genes to be significantly differentially spliced and of those 311 were not found to be differentially expressed at the gene level, indicating that alternative splicing is as important alternative mechanism to gene expression differences. 59 novel splice sites were identified, as well as 152 known splice sites and 218 exon significant differential usage with a p-value of < 0.05. The genes with most significant levels of alternative splicing and found by more than one approach were DYNLL1, TMEM14C, CRNDE, BRD4 and BCL2L1, several of which are also seen in other cancers with mutated SF3B1. Conclusions: Hotspot mutations in SF3B1 result in alternative splicing of genes as well as the introduction of novel splice sites. The confirmation that SF3B1 hotspot mutations in MM increases alternative splicing as well as the identification of the genes undergoing alternative splicing may present novel therapeutic targets. Gene expression analysis of these samples identifies key deregulated pathways, perhaps in response to alternative splicing, including the UPR and protein secretion pathways. These analyses indicate that disruption of these pathways are potential avenues of therapeutic intervention in patients with SF3B1 mutations. Disclosures Ortiz: Celgene Corporation: Employment, Equity Ownership. Flynt:Celgene Corporation: Employment, Equity Ownership. Thakurta:Celgene Corporation: Employment, Equity Ownership. Morgan:Celgene: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria; Janssen: Research Funding; Bristol-Myers Squibb: Consultancy, Honoraria.

scholarly journals Integrated Analysis of Global DNA Methylation and Transcription Reveals a Leukemia Subtype with Extreme Hypermethylation Associated with Silencing of Regulators of Differentiation

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2608-2608
Author(s):  
Claudia Gebhard ◽  
Roger Mulet-Lazaro ◽  
Lucia Schwarzfischer ◽  
Dagmar Glatz ◽  
Margit Nuetzel ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Promoter Methylation ◽  
Research Funding ◽  
Promoter Hypermethylation ◽  
Ctcf Binding ◽  
P Value ◽  
Global Dna Methylation ◽  
Employment Equity ◽  
Equity Ownership

Abstract Acute myeloid leukemia (AML) represents a highly heterogeneous myeloid stem cell disorder classified based on various genetic defects. Besides genetic alterations, epigenetic changes are recognized as an additional mechanism contributing to leukemogenesis, but insight into the latter process remains minor. Using a combination of Methyl-CpG-Immunoprecipitation (MCIp-chip) and MALDI-TOF analysis of bisulfite-treated DNA in a cohort of 196 AML patients we previously demonstrated that (cyto)genetically defined AML subtypes, including CBFB-MYH11, AML-ETO, NPM1-mut, CEBPA-mut or IDH1/2-mut subtypes, express specific DNA-methylation profiles (Gebhard et al, Leukemia, 2018). A fraction of AML patients (5/196) displayed a unique abnormal hypermethylation profile that was completely distinct from any other AML subtype. These patients present immature leukemia (FAB M0, M1) with various chromosomal aberrations but very few mutations (e.g. no IDH1/2, KRAS, DNMT3A) that might explain the CpG island methylator phenotype (CIMP) phenotype. The CIMP patients showed high resemblance with a recently reported CEBPA methylated subgroup (Wouters et al, 2007 and Figueroa et al, 2009), which we confirmed by MCIp-chip and MALDI-TOF analysis. To explore the whole range of epigenetic alterations in the CIMP-AML patients we performed in-depth global DNA methylation and gene expression analyses (MCIp-seq and RNA-seq) in 45 AML and 12 CIMP patients from both studies. Principle component analysis and t-distributed stochastic neighbor embedding (t-SNE) revealed that CIMP patients express a unique DNA-methylation and gene-expression signature that separated them from all other AMLs. We could discriminate promoter methylation from non-promoter methylation by selecting MCIp-seq peaks within 3kb around TSS. Promoter hypermethylation was highly associated with repression of genes (PCC = -0.053, p-value = 0.00075). Hypermethylation of non-promoter regions was more strongly associated with upregulation of genes (PCC = 0.046, p-value = 4.613e-06). Interestingly, differentially methylated regions also showed a positive association with myeloid lineage CTCF binding sites (27% vs 18% expected, p-value < 2.2e-16 in a chi-square test of independence). Methylation of CTCF sites causes loss of CTCF binding, which has been reported to disrupt boundaries between so-called topologically associated domains (TADs), allowing enhancers located in a particular TAD to become accessible to genes in adjacent TADs and affect their transcription. Whether this is the case is under investigation. In this study we particularly focused on the role of hypermethylation of promoters in CIMP-AMLs. Promoters of many transcriptional regulators that are involved in the differentiation of myeloid lineages of which several are frequently mutated in AML were hypermethylated and repressed, including CEBPA, CEBPD, IRF8, GATA2, KLF4, MITF or MAFB. Notably, HMGA2, a critical regulator of myeloid progenitor expansion, exhibited the largest degree of CIMP promoter hypermethylation compared to the other AMLs, accompanied by a reduction in gene expression. Moreover, multiple members of the HOXB family and KLF1 (erythroid differentiation) were methylated and repressed as well. In addition, these patients frequently showed hypermethylation of many chromatin factors (e.g. LMNA, CHD7 or TET2). Hypermethylation of the TET2 promoter could result in a loss of maintenance DNA demethylation and therefore successive hypermethylation at CpG islands. We carried out regulome-capture-bisulfite sequencing on CIMP-AMLs compared to other AML samples and normal blood cell controls and confirmed methylation of the same transcription and chromatin factor promoters. We conclude that these leukemias represent very primitive HSCPs which are blocked in differentiation into multiple hematopoietic lineages, due to the absence of regulators of these lineages. Although the underlying cause for the extreme hypermethylation signature is still subject to ongoing studies, the consequence of promoter hypermethylation is silencing of key lineage regulators causing the differentiation arrest in these cells. We argue that these patients may particularly benefit from therapies that revert DNA methylation. Disclosures Ehninger: Cellex Gesellschaft fuer Zellgewinnung mbH: Employment, Equity Ownership; GEMoaB Monoclonals GmbH: Employment, Equity Ownership; Bayer: Research Funding. Thiede:AgenDix: Other: Ownership; Novartis: Honoraria, Research Funding.

Phase II Study of the Cyclin-Dependent Kinase (CDK) Inhibitor Dinaciclib (SCH 727965) In Patients with Advanced Acute Leukemias

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3287-3287 ◽  
Author(s):  
Ivana Gojo ◽  
Alison Walker ◽  
Maureen Cooper ◽  
Eric J Feldman ◽  
Swaminathan Padmanabhan ◽  
...  
Keyword(s):  
Research Funding ◽  
Post Treatment ◽  
Parp Cleavage ◽  
Employment Equity ◽  
Acute Leukemias ◽  
Early Management ◽  
Tumor Lysis ◽  
Equity Ownership ◽  
Pre Treatment

Abstract Abstract 3287 Background: Dinaciclib is a potent and selective inhibitor of the CDKs 1, 2, 5, and 9 that has demonstrated anti-tumor activity against both myeloid and lymphoid leukemia cell lines in vitro and human tumor xenografts in vivo. Methods: A randomized, multicenter, open-label phase 2 study of dinaciclib 50 mg/m2 administered by 2-hour i.v. infusion once every 21 days was initiated with the goal of assessing its efficacy and safety in patients (pts) with advanced acute myeloid (AML, ≥60 years old) or lymphoid (ALL, ≥18 years old) leukemia. AML pts were randomized between dinaciclib and gemtuzumab ozogamicin (GO) with cross-over to dinaciclib if no response to GO, while ALL pts only received dinaciclib. Intra-patient dose escalation of dinaciclib to 70 mg/m2 in cycle 2 was allowed. Twenty-six pts were treated on study (20 AML, 6 ALL). Data on 14 AML (2 cross-over from GO) and 6 ALL pts treated with dinaciclib are presented. Their median age was 70 (range 38–76) years and 70% were male. Sixteen pts were refractory and 4 pts had relapsed after a median of one (range 1–4) chemotherapy regimens. Four AML pts had complex karyotypes (≥3 abnormalities), 2 monosomy 7, 2 trisomy 8, 1 der (1:7)(q10;p10), 1 trisomy 21, 1 deletion 9q, and 3 had normal karyotype. Two ALL pts had t(9;22). Response: Anti-leukemia activity was observed in 60% of pts. Ten of 13 pts with circulating blasts (7/7 AML and 3/6 ALL) had >50% and 6 pts (4 AML, 2 ALL) >80% decrease in the absolute blast count (ABC) within 24 hours of the first dinaciclib dose. An additional pt had a 29% decrease in ABC. The median pre-treatment ABC was 1085 (range 220–9975) and the median ABC nadir was 169 (range 0–1350). The median duration of blast nadir was 6 days (range 2–23). A representative graph from an AML patient (below) shows a rapid decrease of circulating blasts and WBC after treatment, followed by a gradual recovery. Two patients had >50% reduction of marrow blasts (35% on d1 to 17% on d 42 in an AML pt; 81% on d1 to 27% on d 21 in an ALL pt). However, no objective responses by International Working Group criteria were observed. The median number of treatment cycles was 1 (range 1–5), with 10 pts receiving more than one cycle of treatment. Eight pts were treated with dinaciclib 70 mg/m2 starting in cycle 2. Toxicity: Treatment related AE's occurring in >30% of pts included diarrhea, nausea, vomiting, anemia, elevated AST, fatigue, leukopenia, hypocalcemia, and hypotension. The most common CTCAE v3 treatment-related grade 3 and 4 toxicities, occurring in 3 or more pts, were anemia, leukopenia, febrile neutropenia, thrombocytopenia, fatigue, increased AST, and tumor lysis syndrome (TLS). Laboratory evidence of tumor lysis in cycle 1, using the Cairo-Bishop criteria, was seen in 6 pts in addition to 3 pts with clinical TLS (JCO 2008;26:2767). Hyperacute TLS requiring hemodialysis occurred in one pt with AML, who died of acute renal failure. Subsequently, all pts were aggressively managed to prevent and treat TLS (hospitalization, hydration, allopurinol, rasburicase, oral phosphate binder administration, and early management of hyperkalemia). An additional 9 pts died on study, 8 pts from leukemia progression and 1 pt from intracranial bleed due to disease-related thrombocytopenia. Pharmacodynamics: Pre-treatment, 4 and 24 hrs post end-of-infusion samples of circulating leukemic blasts were obtained from 1 AML and 3 ALL pts. By Western blot, post-treatment decrease in Mcl-1 and increase in PARP cleavage were seen in all 4 pts at 4 hrs post-treatment, confirming that in vivo inhibition of CDKs was achieved, but recovery of Mcl-1 at 24 hrs was observed in all 4 pts, suggesting that inhibition was lost at 24 hrs. Decline in p-Rb was observed in 1 pt, while 2 pts had almost undetectable p-Rb levels at baseline. Conclusion: Dinaciclib showed anti-leukemia activity in this heavily pre-treated patient population. TLS was a notable toxicity, but was manageable in most pts with aggressive prophylaxis, monitoring and treatment. Early blast recovery and short duration of nadir observed on this study, combined with PK data showing a short t1/2 (1.5-3.3 hours) for dinaciclib and PD data demonstrating rapid reexpression of Mcl-1, support either use of longer infusion schedules (currently explored in solid tumors) or more frequent drug administration. Further exploration of dinaciclib dose and schedules in AML and ALL is planned. Disclosures: Gojo: Merck & Co.: Research Funding. Off Label Use: SCH 727965 (dinaciclib) is an investigational drug. Padmanabhan:Schering-Plough: Consultancy; Merck & Co.: Research Funding. Small:Merck & Co.: Employment, Equity Ownership. Zhang:Merck & Co.: Employment. Sadowska:Merck & Co.: Research Funding. Bannerji:Merck & Co.: Employment, Equity Ownership.

Gene Risk Scores Based on Expression of 6 Genes Quanitated by Nuclease Protection Assay in Formalin Fixed Paraffin-Embedded Tissue (FFPET) Specimens From CHOP and RCHOP Treated Patients with Diffuse Large B-Cell Lymphoma (DLBCL) Predict Outcome: An ECOG and SWOG Study

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 87-87
Author(s):  
Jane N. Winter ◽  
Fangxin Hong ◽  
Lisa M. Rimsza ◽  
Michael LeBlanc ◽  
Daina Variakojis ◽  
...  
Keyword(s):  
Gene Expression ◽  
Clinical Outcome ◽  
Predictive Model ◽  
Risk Scores ◽  
Protection Assay ◽  
Employment Equity ◽  
Nuclease Protection Assay ◽  
Equity Ownership ◽  
Correlative Studies ◽  
Molecular Predictor

Abstract Abstract 87 Background: The International Prognostic Index (IPI) remains the most powerful predictor of clinical outcome in DLBCL in the R-CHOP era, serving as a useful surrogate for the biology we are just beginning to understand. With a follow-up of more than 9 years, its uniformly staged and treated patients and its prospectively collected unstained slides for correlative studies, the clinical data set from E4494, the US Intergroup trial comparing CHOP and RCHOP in patients >60 years with DLBCL, stands as a valuable resource for investigating prognosis in DLBCL. Based on immunohistochemistry, we previously showed that rituximab modulates the prognostic significance of some biomarkers in DLBCL. With these same specimens, the quantitative nuclease protection assay (qNPA), a methodology for measuring mRNA levels in FFPET, was used to develop separate prognostic signatures for the CHOP and RCHOP arms that can be simply applied using stored unstained slides. Methods: Five micron unstained FFPET sections from 183 eligible and evaluable cases enrolled on E4494 and submitted for prospective immunohistochemical correlative studies more than ten years ago were used for this analysis. Tissue was scraped from slides and a multiplexed qNPA was performed in triplicate using a customized Array Plate assay (HTG, Inc) for 43 genes of interest. TBP served as a housekeeping gene. Association between standardized log gene expression and patient failure-free survival (FFS) and overall survival (OS) was obtained using the Cox proportional hazards model. A weighted analysis was used to eliminate the confounding effect of maintenance rituximab. Genes that showed at least marginal significance in the univariate analysis were used to perform LASSO (penalized method to select best subset) to select a final list of genes in the multivariate analysis. Using the predictive model for either CHOP or R-CHOP induction, individual risk scores were calculated based on the multivariable model, and cases were dichotomized into low and high risk groups based on the median risk score. The model was then validated using the Lenz dataset (NEJM, 2008). Results: In six cases, tissue from slides prepared >10 years ago was compared to freshly cut sections from corresponding blocks, and showed excellent concordance (corr=0.86). On-study characteristics for the 176 cases with analyzable data were representative of the greater E4494 patient population. Six gene predictors were developed for each arm of the trial: RCHOP: FN1, LMO2, AKT1, HIF1a, AKT3, BCL2; and CHOP: PDCD4, HLADRB1, COL3A1, LMO2, ROBO4, TP53. Both signatures proved powerful predictors of FFS and OS among CHOP (FFS: p=0.0031; OS: p=0.0013) and RCHOP (FFS: p=0.001; OS=p=0.0015) treated patients. When adjusted for the clinically-based IPI, the gene-risk score retained its significance (CHOP: FFS p=0.0007; OS p=0.0011; RCHOP: FFS p=0.003; OS, p=0.001) while the IPI became only marginally significant (CHOP: FFS: p=0.06, OS p=0.06; R-CHOP: FFS p=0.09, OS p=0.02), suggesting that the gene predictor accounted for much of the predictive power of the IPI. The predictive model was then validated using the Lenz dataset for OS. The predictor models for CHOP and R-CHOP-treated patients effectively dichotomized patients into prognostic subgroups (CHOP: p<0.0001; RCHOP: p=0.0014; see figure below) and this difference was maintained when the subset over age 60 was analyzed (CHOP: p=0.0008; RCHOP: p=0.017). When adjusted for the IPI, the molecular predictor developed for CHOP treated patients remained robust (OS: P=.0001; HR 2.39), while the molecular predictor for RCHOP predicted OS marginally (p=.06; HR 1.82), with shorter followup than the CHOP cohort. Conclusions: Unstained slides from FFPET stored for many years may be used to investigate gene expression in lymphoma biopsy specimens for which there is mature followup. Gene risk scores based on the expression of a limited number of genes are powerful predictors of clinical outcome. Disclosures: Pollock: HTG, Inc.: Employment, Equity Ownership. Botros:HTG, Inc.: Employment, Equity Ownership. Horning:Genentech: Employment, Equity Ownership.

Mechanistic Insights Into Fetal Hemoglobin (HbF) Induction Through Chemical Inhibition Of Histone Deacetylase 1 and 2 (HDAC1/2)

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2253-2253 ◽  
Author(s):  
Jeffrey R Shearstone ◽  
John H van Duzer ◽  
Simon S Jones ◽  
Matthew Jarpe
Keyword(s):  
Gene Expression ◽  
Histone Deacetylase ◽  
Globin Gene ◽  
Gene Set Enrichment Analysis ◽  
Binding Motif ◽  
Nurd Complex ◽  
Employment Equity ◽  
Nucleosome Remodeling ◽  
Genetic Ablation ◽  
Equity Ownership

Abstract Induction of HbF is an established therapeutic strategy for the treatment of sickle cell disease (SCD), and could also be effective in treating beta-thalassemia (bT). Fetal beta-like globin gene (HbG) expression is silenced in adults partly through the nucleosome remodeling and histone deacetylase (NuRD) complex, which contains HDAC1/2 (Sankaran VG, Science, 2008). Genetic ablation of HDAC1 or HDAC2, but not HDAC3, results in the induction of HbG expression (Bradner JE, Proc Natl Acad Sci, 2010). Furthermore, we have previously shown that selective chemical inhibitors of HDAC1 and 2 elicit a dose and time dependent induction of HbG mRNA and HbF protein in cultured human CD34+ bone marrow cells undergoing erythroid differentiation (Shearstone JS, ASH Annual Meeting Abstracts, 2012). However, the mechanism through which HDAC1/2 inhibition leads to activation of HbG remains largely unknown. In this work, we have utilized our proof of concept molecule, ACY-957, to investigate changes in gene expression and chromatin organization that result from inhibition of HDAC1/2. Gene expression profiling was performed on cells treated with ACY-957 (n=3) or vehicle (n=3) using Affymetrix PrimeView GeneChips. Treatment of early erythroblasts (CD71+, GlyA-) resulted in the up and down regulation of 1294 and 681 transcript probe sets, respectively. In comparison, treatment of late erythroblasts (CD71+, GlyA+) resulted in a total of 255 transcript probe set changes. This finding is consistent with follow-up experiments demonstrating that ACY-957 is unable to induce HbG in cells positive for both CD71 and GlyA. Taken together, these results suggest that erythroblasts become less responsive to HDAC inhibition as they mature. Gene set enrichment analysis using public domain data revealed that genes up- or down-regulated by HDAC1/2 shRNA knockdown are significantly overrepresented in the list of genes induced or repressed by ACY-957, respectively; suggesting pharmacologic inhibition of HDAC1/2 recapitulates genetic ablation. We also identified significant enrichment in other gene sets involving targets linked to HbG regulation, including lysine-specific demethylase 1 (LSD1) (Shi L, Nature Medicine, 2012). GeneChip and quantitative real-time PCR time course experiments show ACY-957 treatment leads to a decrease in Bcl11A (2-fold) and Sox6 (10-fold) mRNA, known repressors of fetal globin synthesis, and an increase in Klf2 (2-fold) and Gata2 (8-fold) mRNA, proposed fetal globin activators. This result is consistent with work by others that show Gata2 is suppressed, in part, by the NuRD complex (Hong W, EMBO Journal, 2005) and that Gata2 binding at the HbG promoter leads to increased levels of HbG expression (Zhu X, PLoS One, 2012). Interestingly, Gata2 induction preceded Sox6 suppression in ACY-957 treated cells and the Sox6 promoter contains 8 canonical WGATAR binding sites and one Gata2-specific binding motif, raising the possibility suppression of Sox6 by ACY-957 is mediated by Gata2 induction. To investigate these possibilities, we have performed chromatin immunoprecipitation coupled with next generation sequencing (ChIP-seq) for HDAC1, HDAC2, Gata2, and the HDAC2-specific histone modification H3K56 in ACY-957 and vehicle treated cells. These experiments will be discussed. ChIP-seq data, both by itself and in combination with gene expression data, will provide further insight into the mechanism through which HDAC1/2 regulates HbF synthesis. Disclosures: Shearstone: Acetylon Pharmaceuticals, Inc.: Employment, Equity Ownership. van Duzer:Acetylon Pharmaceuticals, Inc.: Employment, Equity Ownership. Jones:Acetylon Pharmaceuticals, Inc: Employment, Equity Ownership. Jarpe:Acetylon Pharmaceuticals, Inc.: Employment, Equity Ownership.

DOT1L Inhibitor EPZ-5676 Displays Synergistic Antiproliferative Activity in Combination with Standard of Care Drugs or DNA Hypomethylating Agents in MLL-Rearranged Leukemia Cells

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3930-3930 ◽  
Author(s):  
Christine R Klaus ◽  
Scott R. Daigle ◽  
Dorothy Iwanowics ◽  
L. Danielle Johnston ◽  
Carly A Therkelsen ◽  
...  
Keyword(s):  
Cell Line ◽  
Stock Options ◽  
Leukemia Cell ◽  
Standard Of Care ◽  
Treatment Model ◽  
Hypomethylating Agents ◽  
Employment Equity ◽  
Acute Leukemias ◽  
Equity Ownership ◽  
Pre Treatment

Abstract EPZ-5676 is a small molecule inhibitor of the histone methyltransferase DOT1L that is currently under clinical investigation as a potential therapy for acute leukemias bearing MLL-rearrangements. Gene knockout and small molecule inhibitor studies have demonstrated that DOT1L is required for MLL-fusion protein–mediated leukemogenesis in model systems. In preclinical studies EPZ-5676 promoted cell killing of acute leukemia lines bearing MLL translocations in vitro while sparing those without MLL gene translocations and also caused sustained tumor regressions in a rat xenograft model of MLL-rearranged leukemia [Daigle et al. Blood 2013]. To support potential future clinical scenarios, we evaluated the activity of EPZ-5676 in combination with current standard of care agents for acute leukemias as well as other chromatin modifying drugs in cell proliferation assays with three human acute leukemia cell lines; Molm-13 (MLL-AF9 expressing acute myeloid leukemia (AML)), MV4-11 (MLL-AF4 expressing acute biphenotypic leukemia cell line) and SKM-1 (non-MLL-rearranged AML). We established a high density combination platform suitable for testing the anti-proliferative activity of a complete titration matrix of two agents with multiple replicate points to enable generation of statistically meaningful results. This platform was used to evaluate the anti-proliferative effects of EPZ-5676 combinations tested in a co-treatment model in which the second agent was added along with EPZ-5676 at the beginning of the assay, or in a pre-treatment model in which cells were incubated for several days in the presence of EPZ-5676 prior to the addition of the second agent. The drug combination analysis was performed using the Chou-Talalay method [Chou TC Pharmacological Reviews 2006]. Graphs representing values of combination index (CI) versus Fractional effect (Fa) known as Fa-CI plots were generated and synergy was evaluated. Drug synergy was statistically defined by CI values less than 1, antagonism by CI >1 and additive effect by CI equal to 1. We found that EPZ-5676 acts synergistically with the AML standard of care agents cytarabine or daunorubicin in Molm-13 and MV4-11 MLL-rearranged cell lines. However, in the non-rearranged SKM-1 cell line EPZ-5676 had no effect alone and did not act synergistically with cytarabine or daunorubicin. Moreover, a persistent combination benefit was observed even when EPZ-5676 was washed out prior to the addition of the standard of care agents (Figure 1), suggesting that EPZ-5676 sets up a durable altered chromatin state that enhances the effect of chemotherapeutic agents in MLL-rearranged cells. We are currently exploring the mechanism of action of this synergy in more detail.Figure 1. Fa-CI plots show that EPZ-5676 and cytarabine act synergistically to induce an antiproliferative effect in the Molm-13 cell line in a pre-treatment model. (A) Ten-day continuous dosing of EPZ-5676 with addition of cytarabine at day 7 showed a range of fractional effects with CI values <1 denoting synergy. (B) EPZ-5676 was removed at day 7 prior to the addition of cytarabine showing durable combination benefit.Figure 1. Fa-CI plots show that EPZ-5676 and cytarabine act synergistically to induce an antiproliferative effect in the Molm-13 cell line in a pre-treatment model. (A) Ten-day continuous dosing of EPZ-5676 with addition of cytarabine at day 7 showed a range of fractional effects with CI values <1 denoting synergy. (B) EPZ-5676 was removed at day 7 prior to the addition of cytarabine showing durable combination benefit. Our evaluation of EPZ-5676 in conjunction with other chromatin modifying drugs also revealed a consistent combination benefit including synergy with DNA hypomethylating agents. In summary, our results indicate that EPZ-5676 is highly efficacious as a single agent and is synergistic with other anticancer agents including AML standard of care drugs and DNA hypomethylating agents in MLL-rearranged cells. Disclosures: Klaus: Epizyme, Inc.: Employment, Equity Ownership, Patents & Royalties, Stock Options Other. Daigle:Epizyme, Inc.: Employment, Equity Ownership, Patents & Royalties, Stock Options Other. Iwanowics:Epizyme, Inc.: Employment, Equity Ownership, Stock Options Other. Johnston:Epizyme, Inc: Employment, Equity Ownership, Stock Options Other. Therkelsen:Epizyme, Inc.: Employment, Equity Ownership, Stock Options Other. Smith:Epizyme, Inc.: Employment, Equity Ownership, Stock Options Other. Moyer:Epizyme, Inc.: Employment, Equity Ownership, Stock Options Other. Copeland:Epizyme Inc. : Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties; Mersana: Membership on an entity’s Board of Directors or advisory committees. Olhava:Epizyme, Inc: Employment, Equity Ownership, Patents & Royalties, Stock Options Other. Porter Scott:Epizyme, Inc: Employment, Equity Ownership, Patents & Royalties, Stock Options Other. Pollock:Epizyme Inc.: Employment, Equity Ownership, Patents & Royalties, Stock Options Other. Raimondi:Epizyme, Inc: Employment, Equity Ownership, Patents & Royalties, Stock Options Other.

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