Diverse and Targetable Kinase Alterations Drive Histiocytic Neoplasms

Abstract Histiocytic neoplasms are clonal, hematopoietic disorders characterized by an accumulation of abnormal monocyte-derived dendritic cells or macrophages in Langerhans Cell (LCH) and non-Langerhans (non-LCH) histiocytoses, respectively. The discovery of the BRAF V600E mutation in ~50% of patients with LCH and the non-LCH Erdheim-Chester Disease (ECD) provided the first molecular target in these patients and novel insights into the pathogenesis of these disorders. However, recurrent mutations in the majority of the ~50% of BRAF V600E-wild type patients with non-LCH are unknown. Moreover, recurrent mutations outside of the MAP kinase pathway are undefined throughout histiocytic neoplasms. To address these issues, we performed whole exome sequencing (WES) of frozen biopsies from 24 patients with LCH (n=10) or ECD (n=14) paired with peripheral blood mononuclear cells. 13/24 patients also underwent RNA sequencing (RNA-seq). All mutations in activating kinases were validated by droplet-digital PCR, while targeted-capture next-generation sequencing validated all others. Both adult (n=18; n=2 with LCH) and pediatric cases (n=9; n=8 with LCH) were included. Using combined WES/RNA-seq, activating kinase alterations were identified in 100% of patients. In LCH, 60% and 40% had BRAF V600E and MAP2K1 mutations, respectively. In non-LCH 51%, 14%, 14%, and 7% were BRAFV600E, ARAF, MAP2K1, and NRAS mutant (Fig1A). Overall, a mean of 7 non-synonymous mutations per adult patient was identified (range 1-22) compared with 5 mutations per pediatric patient (range 4-9; p =ns). Mutations affecting diverse cellular processes were found to co-exist with kinase mutations including mutations in epigenetic modifiers and the p38/MAPK pathway. In addition to kinase point mutations, RNA-seq identified recurrent, in-frame kinase fusions-a first for these disorders. All identified fusions were validated using FISH and RT-PCR. This includes novel fusions in BRAF (RNF11-BRAF and CLIP2-BRAF), as well as therapeutically important fusions in ALK (2 separate KIF5B-ALK fusions) and NTRK1 (LMNA-NTRK1;Fig1B). Expression of each fusion in Ba/F3 cells conferred cytokine-independent growth. Importantly, the BRAF fusions were found to be sensitive to MEK inhibition but resistant to vemurafenib while the ALK fusions conferred sensitivity to the ALK inhibitors crizotinib or alectinib. We next interrogated a validation cohort of 37 BRAF V600E-wild type, non-LCH, formalin-fixed, paraffin-embedded tissue samples using targeted mutational profiling for MAP2K1, ARAF, NRAS, KRAS, and PIK3CA. This revealed activating mutations in MAP2K1 (32%; n=12), NRAS (16%; n=6), KRAS (11%; n=4), PIK3CA (8%; n=3), and ARAF (3%; n=1). Three of the investigated non-LCH patients with refractory disease and progressive organ dysfunction were treated with targeted therapies based on the discovery of novel kinase alterations described above. Treatment of 2 refractory MAP2K1- mutant, non-LCH patients with MEK inhibitors (trametinib or cobimetinib) resulted in dramatic clinical improvement (Fig1C). Both patients have been maintained on MEK inhibitor single-agent therapy with a sustained clinical response for >100 days. Further evidence of effective targeted inhibition was found in a refractory ECD patient carrying an ARAF S214A mutation. This patient failed to respond to 3 lines of prior therapies and suffered near blindness due to disease infiltration in the retina and optic nerves. Given a recent report of complete response to sorafenib in a lung cancer patient with an ARAF S214C mutation, we initiated sorafenib. Within 12 weeks, there was improvement in the patientÕs eyesight and decreased infiltrative disease, coinciding with >50% decrease in mutant ARAF DNA in plasma cell-free DNA. Whole exome and transcriptome sequencing identified activating kinase mutations or translocations in all patients with the common downstream effect of activating the MAPK pathway. The preliminary, dramatic, clinical efficacy observed with use of MEK and RAF inhibitors in MAP2K1 - and ARAF-mutated, non-LCH patients further supports the central role of targeting the MAPK pathway in these tumors. The discovery of the discussed mutations and fusions in diverse kinases provides critical new insights into the genetic events central to a spectrum of adult and pediatric histiocytic neoplasms. Figure 1. Figure 1. Disclosures Off Label Use: This abstract describes use of MEK inhibitors (both tremetinib and cobimetinib) as well as sorafenib for MEK1 and ARAF mutant histiocytosis. . Stephens:Foundation Medicine, Inc.: Employment, Equity Ownership. Miller:Foundation Medicine, Inc.: Employment, Equity Ownership. Ross:Foundation Medicine Inc.: Employment. Ali:Foundation Medicine Inc.: Employment. Hyman:Chugai Pharma: Consultancy; Biotherapeutics: Consultancy; Atara: Consultancy, Honoraria.

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Recurrent Mutations of Multiple Components of Cohesin Complex in Myeloid Neoplasms

Blood ◽

10.1182/blood.v120.21.782.782 ◽

2012 ◽

Vol 120 (21) ◽

pp. 782-782

Author(s):

Ayana Kon ◽

Lee-Yung Shih ◽

Masashi Minamino ◽

Masashi Sanada ◽

Yuichi Shiraishi ◽

...

Keyword(s):

Gene Expression ◽

Cell Lines ◽

Gene Mutations ◽

Cohesin Complex ◽

Wild Type ◽

Myeloid Neoplasms ◽

Whole Exome ◽

Recurrent Mutations ◽

Equity Ownership

Abstract Abstract 782 Recent genetic studies have revealed a number of novel gene mutations in myeloid malignancies, unmasking an unexpected role of deregulated histone modification and DNA methylation in both acute and chronic myeloid neoplasms. However, our knowledge about the spectrum of gene mutations in myeloid neoplasms is still incomplete. In the previous study, we analyzed 29 paired tumor-normal samples with chronic myeloid neoplasms with myelodysplastic features using whole exome sequencing (Yoshida et al., Nature 2011). Although the major discovery was frequent spliceosome mutations tightly associated with myelodysplasia phenotypes, hundreds of unreported gene mutations were also identified, among which we identified recurrent mutations involving STAG2, a core cohesin component, and also two other cohesin components, including STAG1 and PDS5B. Cohesin is a multimeric protein complex conserved across species and is composed of four core subunits, i.e., SMC1, SMC3, RAD21 and STAG proteins, together with several regulatory proteins. Forming a ring-like structure, cohesin is engaged in cohesion of sister chromatids in mitosis, post-replicative DNA repair and regulation of gene expression. To investigate a possible role of cohesin mutations in myeloid leukemogenesis, an additional 534 primary specimens of various myeloid neoplasms was examined for mutations in a total of 9 components of the cohesin and related complexes, using high-throughput sequencing. Copy number alterations in cohesin loci were also interrogated by SNP arrays. In total, 58 mutations and 19 deletions were confirmed by Sanger sequencing in 73 out of 563 primary myeloid neoplasms (13%). Mutations/deletions were found in a variety of myeloid neoplasms, including AML (22/131), CMML (15/86), MDS (26/205) and CML (8/65), with much lower mutation frequencies in MPN (2/76), largely in a mutually exclusive manner. In MDS, mutations were more frequent in RCMD and RAEB (19.5%) but rare in RA, RARS, RCMD-RS and 5q- syndrome (3.4%). Cohesin mutations were significantly associated with poor prognosis in CMML, but not in MDS cases. Cohesin mutations frequently coexisted with other common mutations in myeloid neoplasms, significantly associated with spliceosome mutations. Deep sequencing of these mutant alleles was performed in 19 cases with cohesin mutations. Majority of the cohesin mutations (16/19) existed in the major tumor populations, indicating their early origin during leukemogenesis. Next, we investigated a possible impact of mutations on cohesin functions, where 17 myeloid leukemia cell lines with or without cohesin mutations were examined for expression of each cohesin component and their chromatin-bound fractions. Interestingly, the chromatin-bound fraction of one or more components of cohesin was substantially reduced in cell lines having mutated or defective cohesin components, suggesting substantial loss of cohesin-bound sites on chromatin. Finally, we examined the effect of forced expression of wild-type cohesin on cell proliferation of cohesin-defective cells. Introduction of the wild-type RAD21 and STAG2 suppressed the cell growth of RAD21- (Kasumi-1 and MOLM13) and STAG2-defective (MOLM13) cell lines, respectively, supporting a leukemogenic role of compromised cohesin functions. Less frequent mutations of cohesin components have been described in other cancers, where impaired cohesion and consequent aneuploidy were implicated in oncogenic action. However, 23 cohesin-mutated cases of our cohort had completely normal karyotypes, suggesting that cohesin-mutated cells were not clonally selected because of aneuploidy. Alternatively, a growing body of evidence suggests that cohesin regulate gene expression, arguing for the possibility that cohesin mutations might participate in leukemogenesis through deregulated gene expression. Of additional note, the number of non-silent mutations determined by our whole exome analysis was significantly higher in 6 cohesin-mutated cases compared to non-mutated cases. Since cohesin also participates in post-replicative DNA repair, this may suggest that compromised cohesin function could induce DNA hypermutability and contribute to leukemogenesis. In conclusion, we report a new class of common genetic targets in myeloid malignancies, the cohesin complex. Our findings highlight a possible role of compromised cohesin functions in myeloid leukemogenesis. Disclosures: Haferlach: MLL Munich Leukemia Laboratory: Equity Ownership. Alpermann:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership.

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Real-Time Genomic Profiling Identifies Novel Mutations and Improved Therapy for Histiocytoses

Blood ◽

10.1182/blood.v128.22.2723.2723 ◽

2016 ◽

Vol 128 (22) ◽

pp. 2723-2723

Author(s):

Lynn Lee ◽

Anjelika Gasilina ◽

Jayeeta Roychoudhury ◽

Jason Clark ◽

Francis X McCormack ◽

...

Keyword(s):

Real Time ◽

Response To Therapy ◽

Braf V600e ◽

Biological Behavior ◽

Inadequate Response ◽

Genomic Profiling ◽

Employment Equity ◽

Mek Inhibitors ◽

Equity Ownership ◽

Mutant Forms

Abstract BACKGROUND: The histiocytic disorders include several heterogeneous diseases including Langerhans cell histiocytosis (LCH,) juvenile xanthogranuloma, Erdheim-Chester disease (ECD,) and Rosai-Dorfman disease. These conditions have variable clinical courses and can be refractory to contemporary therapy, resulting in end-organ damage or even death. The etiopathogenesis of LCH and the other histiocytoses remained unclear for decades, until the identification of recurring BRAF V600E mutations, and more recently mutations in the downstream gene MAP2K1 (encoding the enzyme MEK) in LCH and ECD. Given the high risk of recurrence and the unpredictable response to therapy in some patients, we sought to characterize the genomic landscape of histiocytic lesions in patients in real-time. Our principal goal was to select alternative treatments for patients with inadequate response to standard therapies. As a secondary goal, we aimed to further characterize the biological effects of MAP2K1 mutations found in LCH and ECD, and determine their susceptibility to targeted therapies. METHODS AND RESULTS: We used a hybrid capture-based sequencing platform to molecularly profile eighty-five patient samples from patients with one of the above diagnoses. Fifteen patient samples (18%) harbored the BRAF V600E point mutation, and four LCH patients carried a novel 5 or 6 amino-acid in-frame deletion (indel) in BRAF (N486_P490del or N486_T491>K.) Eleven patient samples (13%) harbored activating mutations in MAP2K1. Additional recurrently altered genes included NRAS, KRAS and CDKN2A/B. Transcriptomic profiling also identified several patients with recurrent ALK gene fusions, previously described in other malignancies and recently also identified in histiocytosis not-otherwise-specified. One patient with multisystem LCH with CNS involvement was found to have a BRAF indel. She declined systemic chemotherapy, but agreed to treatment with a targeted agent. Based on the likelihood of resistance to BRAF V600E-specific inhibitors, we started treatment with the MEK inhibitor Trametinib resulting in resolution of disease-associated lymphadenopathy within days, and improvement of CNS symptoms as well. She remains in remission 4 months after the initiation of treatment. In two children, multi-system refractory LCH progressed to secondary HLH (hemophagocytic lymphohistiocytosis). Both demonstrated the presence of BRAF-V600E and their disease promptly responded to the BRAF inhibitor Dabrafenib. We then characterized the biological behavior of the MAP2K1 mutations using retroviral transduction in order to stably express these mutations in NIH/3T3 and BaF/3 cells. We demonstrate that these mutations all result in constitutive activation at baseline, as evidenced by increased phosphorylation of the target ERK. These mutant forms of MAP2K1 also express sustained activation of ERK in response to EGF stimulation. Additionally, we tested clinically available MEK inhibitors against mutant forms of MAP2K1, and show that all result in a dose-dependent decrease in phospho-ERK levels in vitro, supporting our hypothesis that MEK inhibition is a valid therapeutic approach in the histiocytic neoplasms. Finally, we demonstrate with an animal model that MAP2K1 is sufficient to induce disease. Using the cre-lox recombinase system in transgenic mice, we selectively express an activated form of MEK in CD11c-positive cells, which is largely restricted to the dendritic cell/macrophage lineage. These mice developed normally, but by a median of 17 weeks of age, mice became moribund and on necropsy exhibited hepatosplenomegaly with extensive infiltration of spleen, liver and lungs with CD68+ macrophages. DISCUSSION: Genomic profiling identified mutations in majority of patients, including a novel BRAF indel. We further show that these mutations result in activation of the MAP kinase pathway, and that activated MAP2K1 is capable of transforming hematopoietic cells resulting in a multisystem histiocytic disorder in mice. Finally, we demonstrate that available MEK inhibitors efficiently block disease-associated mutations. We propose that all patients with histiocytic neoplasms undergo comprehensive genomic profiling in order to identify potential causal mutations, and clinical trials for histiocytoses include MEK inhibitors in relapsed/refractory disease or even as upfront therapy. Disclosures Ali: Foundation Medicine: Employment, Equity Ownership. Bailey:Foundation Medicine, Inc: Employment, Equity Ownership. Stevens:Foundation Medicine Inc.: Employment, Equity Ownership. Ross:Foundation Medicine: Employment, Equity Ownership. Miller:Foundation Medicine: Employment, Equity Ownership.

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WGS and RNA-Seq Is Superior to Conventional Diagnostic Tests in Multiple Myeloma: Ready for Prime Time?

Blood ◽

10.1182/blood-2018-99-113371 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 4442-4442

Author(s):

Alexander Höllein ◽

Sven O. Twardziok ◽

Wencke Walter ◽

Stephan Hutter ◽

Jesús María Hernández ◽

...

Keyword(s):

Multiple Myeloma ◽

Copy Number ◽

Diagnostic Testing ◽

Published Data ◽

Rna Seq ◽

Copy Number Alterations ◽

Structural Variations ◽

Employment Equity ◽

Recurrent Mutations ◽

Equity Ownership

Abstract Background: The diagnosis and risk stratification of multiple myeloma (MM) is based on clinical and cytogenetic tests. Magnetic CD138 enrichment followed by interphase FISH is the gold standard to identify prognostic translocations and copy number alterations. Gene expression (GEP) studies show that MM might consist of various subgroups with distinct clinical outcomes (Zhan et al., Blood 2006). Whole genome sequencing (WGS) and targeted sequencing studies have further shed light onto recurrent mutations in MM and clinical implications are being derived (Lohr et al., Cancer Cell 2014). We set up a single workflow to analyze MM by WGS and RNA-Seq to evaluate whether this diagnostic workup is superior to conventional diagnostic testing. Methods: The cohort comprised 211 patients (pts) diagnosed with MM at our institution from 2011 to 2017. For all pts FISH and WGS was performed on CD138 enriched cells. For WGS 150bp paired-end sequences where generated on Illumina HiseqX and NovaSeq 6000 (Illumina, San Diego, CA). A mixture genomic DNA from multiple anonymous donors was used as normal controls. To remove potential germline variants, each variant was queried against the gnomAD database, variants with global population frequencies >1% where excluded. 47 genes recurrently mutated in MM were selected for evaluation (Kortüm et. al., Blood 2016). Copy number alterations (CNAs) were called using GATK4 and structural variations (SVs) were called using MANTA accounting for missing matched-normal samples. For transcriptome analysis total RNA was sequenced and the resulting estimated gene counts were pre-processed and normalized, applying trimmed mean of M-values normalization method. Results: WGS allowed us to detect 98/102 (96%) translocations that had previously been identified by FISH. Specifically we confirmed 24/24 of t(4;14), 6/7 of t(6;14), 11/12 of t(8;14), 51/53 of t(11;14) and 6/6 of t(14;16) cases by WGS. Moreover, by conventional FISH 12 pts had an IGH (n=4) or MYC (n=8) translocation with an unknown partner chromosome. We identified all 12 translocations by WGS. By WGS we also identified 679/740 (92 %) copy number alterations (CNA) detected by FISH. In detail these were 100/103 del(13q), 17/21 del(17p), 10/10 del(1p) and 79/87 +1q. Concordance rates for trisomies 3, 5, 9, 11, 15 and 19 were 80/91, 75/87, 92/97, 87/97, 53/55 and 86/92, respectively. Zhan et al. defined 7 MM subgroups (CD-1, CD-2, HY, MF, MS, LB, PR) based on GEP. 4 groups (CD-1/CD-2, MF, MS) were genetically defined by recurrent translocations and 1 by hyperploidy (HY). They used 700 probes to separate the groups of which 400 transcripts were recovered in our RNA-Seq analysis (GEPSeq). Supervised clustering grouped all 211 pts at the following frequencies: CD-1 (5%), CD-2 (25%), HY (30%), MF (5%), MS (11%), LB (14%), (PR 10%). 56/62 (90%) of pts that were allocated to CD-1/CD-2 had the characteristic translocation (t(11;14) or t(6;14)), while 23/24 (92%) of pts and 5/10 (50%) pts in GEPSeq group MS and MF had the respective t(4;14) or t(14;16). GEP allocates pts with hyperdiploidy to group HY and 51/63 (81%) pts in HY had a hyperdiploid karyotype by FISH. We specifically queried the WGS data for patients with discrepant FISH and GEPSeq results: WGS identified hyperdiploidy in 10/12 patients that were allocated to HY and could not be assigned by FISH due to insufficient material for complete testing, resulting in a 97% final concordance of GEPSeq and karyotype. One patient in group MS without FISH data for t(4;14) could be confirmed by WGS as harbouring the translocation (concordance 100%). Interestingly in 5/5 patients that were allocated to the MF group by GEPSeq 2 had an IGH-MYC or IGH-MYCN rearrangement respectively and 2 had another IGH rearrangement involving chromosome 8q. By WGS the most frequently mutated (mut) genes were KRAS (26%), NRAS (23 %), TP53 (8%), BRAF (4%) and ATM (2%), which is in line with published data (Lohr 2014). NRASmut was significantly associated with GEPSeq groups CD-2 and HY (p=0.001) and ATMmut with MF, MS and PR (p=0.047). Conclusion: RNA-Seq and WGS prove highly valuable in differentiating genetically distinct MM subgroups. The simultaneous analysis of gene mutations might have future implications for study design and selecting treatment options. A single workflow based on WGS and RNA-Seq provides a comprehensive genetic analysis in MM, is feasible and might substitute conventional diagnostic testing in the near future. Disclosures Höllein: MLL Munich Leukemia Laboratory: Employment. Twardziok:MLL Munich Leukemia Laboratory: Employment. Walter:MLL Munich Leukemia Laboratory: Employment. Hutter:MLL Munich Leukemia Laboratory: Employment. Hernández:MLL Munich Leukemia Laboratory: Employment. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

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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 ◽

10.1182/blood-2019-124337 ◽

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.

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Registered report: COT drives resistance to RAF inhibition through MAP kinase pathway reactivation

eLife ◽

10.7554/elife.11414 ◽

2016 ◽

Vol 5 ◽

Cited By ~ 5

Author(s):

Vidhu Sharma ◽

Lisa Young ◽

Miguel Cavadas ◽

Kate Owen ◽

Keyword(s):

Cancer Biology ◽

Kinase Inhibitor ◽

Mapk Pathway ◽

Single Agent ◽

Braf Inhibitor ◽

Open Science ◽

Mek Inhibitors ◽

Erk Phosphorylation ◽

Erk Activity ◽

A375 Cells

The Reproducibility Project: Cancer Biology seeks to address growing concerns about reproducibility in scientific research by conducting replications of selected experiments from a number of high-profile papers in the field of cancer biology. The papers, which were published between 2010 and 2012, were selected on the basis of citations and Altmetric scores (<xref ref-type="bibr" rid="bib4">Errington et al., 2014</xref>). This Registered Report describes the proposed replication plan of key experiments from “COT drives resistance to RAF inhibition through MAPK pathway reactivation” by Johannessen and colleagues, published in Nature in 2010 (<xref ref-type="bibr" rid="bib10">Johannessen et al., 2010</xref>). The key experiments to be replicated are those reported in Figures 3B, 3D-E, 3I, and 4E-F. In Figures 3B, D-E, RPMI-7951 and OUMS023 cells were reported to exhibit robust ERK/MEK activity concomitant with reduced growth sensitivity in the presence of the BRAF inhibitor PLX4720. MAP3K8 (COT/TPL2) directly regulated MEK/ERK phosphorylation, as the treatment of RPMI-7951 cells with a MAP3K8 kinase inhibitor resulted in a dose-dependent suppression of MEK/ERK activity (Figure 3I). In contrast, MAP3K8-deficient A375 cells remained sensitive to BRAF inhibition, exhibiting reduced growth and MEK/ERK activity during inhibitor treatment. To determine if RAF and MEK inhibitors together can overcome single-agent resistance, MAP3K8-expressing A375 cells treated with PLX4720 along with MEK inhibitors significantly inhibited both cell viability and ERK activation compared to treatment with PLX4720 alone, as reported in Figures 4E-F. The Reproducibility Project: Cancer Biology is collaboration between the Center for Open Science and Science Exchange and the results of the replications will be published in eLife.

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Targeted inhibition of the MAPK pathway: emerging salvage option for progressive life-threatening multisystem LCH

Blood Advances ◽

10.1182/bloodadvances.2016003533 ◽

2017 ◽

Vol 1 (6) ◽

pp. 352-356 ◽

Cited By ~ 11

Author(s):

Alexandra Kolenová ◽

Raphaela Schwentner ◽

Gunhild Jug ◽

Ingrid Simonitsch-Klupp ◽

Christoph Kornauth ◽

...

Keyword(s):

Clinical Response ◽

Clinical Remission ◽

Mapk Pathway ◽

Single Agent ◽

Braf V600e ◽

Longitudinal Assessment ◽

Key Points ◽

Life Threatening ◽

Targeted Inhibition

Key Points Single-agent vemurafenib leads to a rapid and sustained clinical response in severe multisystem LCH but does not eradicate the disease. Longitudinal assessment of BRAF V600E during treatment shows that clinical remission can occur despite significant amounts of mutated BRAF.

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Report of a Phase II Trial of Single-Agent BiTE® Antibody Blinatumomab in Patients with Minimal Residual Disease (MRD) Positive B-Precursor Acute Lymphoblastic Leukemia (ALL).

Blood ◽

10.1182/blood.v114.22.840.840 ◽

2009 ◽

Vol 114 (22) ◽

pp. 840-840 ◽

Cited By ~ 5

Author(s):

Max S Topp ◽

Gerhard Zugmaier ◽

Nicola Goekbuget ◽

Peter Kufer ◽

Mariele Goebeler ◽

...

Keyword(s):

T Cells ◽

Phase Ii ◽

Residual Disease ◽

Lymphoblastic Leukemia ◽

Single Agent ◽

Target Cells ◽

Molecular Response ◽

Intensive Chemotherapy ◽

Employment Equity ◽

Equity Ownership

Abstract Abstract 840 Introduction: In patients with B-precursor ALL, presence of MRD after induction therapy or at any time point later predicts a hematological relapse, despite continued intensive chemotherapy or/and an allogeneic hematological stem cell transplantation (HSCT). Blinatumomab (MT103) targets the CD19 antigen, and is a member of a novel class of bispecific BiTE® antibodies that redirect T cells for lysis of target cells. A phase II study was conducted in collaboration with the German Multicenter Study Group on Adult Lymphoblastic Leukemia (GMALL) in patients with MRD-positive B precursor ALL. Methods: B–precursor ALL patients in complete hematological remission with either persistent or reappeared MRD at any time after consolidation I of front-line therapy were included. One treatment cycle of blinatumomab is a 4-week continuous i.v. infusion, which can be followed by allogeneic HSCT or in case of response by repeated consolidation cycles of blinatumomab with 2-week treatment-free intervals. The dose level at enrollment is 15 μg/m2/day. In patients, who do not respond within four cycles of treatment, the dose can be increased to 30 μg/m2/day. Molecular response is assessed by quantitative PCR of either individual rearrangements of immunoglobulin/TCR-genes or specific genetic aberrations such as bcr/abl or MLL-AF4. Results: Nineteen patients have been treated to date and 16 patients are already evaluable for response. Thirteen of 16 evaluable patients went into molecular complete remission (CR) already after one cycle of blinatumomab. Three patients had a stable MRD level. Of note, 10 of the responding 13 patients had never achieved a molecular CR before blinatumomab treatment despite multiple treatment cycles including tyrosine kinase inhibitors in case of Ph-positive ALL. Two patients in molecular CR had an extra-medullary relapse one in testis and one in cerebro-spinal fluid, both representing immunological niches with limited accessibility for T cells. One patient with stable MRD level had a medullary relapse. All other patients are still relapse-free. None of the patients with molecular CR has shown a medullary relapse to date. The maximum follow-up of molecular CR has been 12 months. Most common adverse events (AEs) included lymphopenia, pyrexia, leucopenia and hypoimmunoglobulinemia. Only one patient had to be discontinued because of a fully reversible epileptical seizure. All other AEs resolved during treatment. Overall, treatment with blinatumomab was well tolerated. Response data of all 21 patients will be presented at ASH. Conclusions: Treatment with blinatumomab converted MRD-positive B–precursor ALL into molecular CR in 13 of 16 evaluable patients with refractory disease as indicated by persistent MRD after intensive chemotherapy. This amounts to a response rate of 81% providing thus the rationale for introducing blinatumomab as a novel agent in the treatment of B–precursor ALL. Disclosures: Zugmaier: Micromet: Employment, Equity Ownership. Goekbuget:Micromet: Consultancy, Research Funding. Kufer:Micromet: Employment, Equity Ownership, Patents & Royalties. Klinger:Micromet: Employment, Equity Ownership. Degenhard:Micromet: Employment, Equity Ownership. Baeuerle:Micromet: Employment, Equity Ownership. Schmidt:Micromet: Employment, Equity Ownership. Nagorsen:Micromet: Employment, Equity Ownership. Riethmueller:Micromet: Consultancy, Equity Ownership. Bargou:Micromet: Consultancy, Equity Ownership, Patents & Royalties.

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Analyses of 81 Chronic Myelomonocytic Leukemia (CMML) for EZH2, TET2, ASXL1, CBL, KRAS, NRAS, RUNX1, IDH1, IDH2, and NPM1 Revealed Mutations In 86.4% of All Patients with TET2 and EZH2 Being of High Prognostic Relevance

Blood ◽

10.1182/blood.v116.21.296.296 ◽

2010 ◽

Vol 116 (21) ◽

pp. 296-296 ◽

Cited By ~ 3

Author(s):

Vera Grossmann ◽

Alexander Kohlmann ◽

Christiane Eder ◽

Nicholas C.P. Cross ◽

Claudia Haferlach ◽

...

Keyword(s):

Candidate Genes ◽

Chronic Myelomonocytic Leukemia ◽

Molecular Heterogeneity ◽

Cell Renewal ◽

Wild Type ◽

Employment Equity ◽

Ras Pathway ◽

Equity Ownership ◽

Exon 4 ◽

Myelomonocytic Leukemia

Abstract Abstract 296 Chronic myelomonocytic leukemia (CMML) is a clonal hematopoietic malignancy that is characterized by features of both a myeloproliferative neoplasm and a myelodysplastic syndrome. Recently, we investigated 81 CMML cases (45 CMML-1, 36 CMML-2) diagnosed between 10/2005 - 9/2008, which had been characterized by chromosome banding analysis and mutation analysis in 6 candidate genes: Mutations were detected in TET2 (44.4%), CBL (22.2%), NRAS (22.2%), KRAS (12.3%), JAK2 (9.8%), RUNX1 (8.7%) (Kohlmann et al., J Clin Oncol. 2010 Jul 19). We now applied amplicon-based deep-sequencing using the small volume Titanium chemistry assay (454 Life Sciences, Branford, CT) to investigate additional 4 candidate genes: IDH1 (exon 4), IDH2 (exon 4) and NPM1 (exon 12) (at known mutational hotspot regions) and the complete coding region of EZH2. EZH2 encodes a catalytic subunit of the polycomb repressive complex 2, a highly conserved histone H3 lysine 27 methyltransferase that influences stem cell renewal. Mutations in EZH2 were recently described to play a role in MPN/MDS. The sequencing library preparation for IDH1, IDH2, NPM1, and EZH2, in total 22 amplicons, was performed using 48.48 Access Array technology (Fluidigm, South San Francisco, CA) to cope with the number of amplicons. In median, 498 reads per amplicon were obtained, thus yielding sufficient coverage for detection of mutations with high sensitivity. After excluding polymorphisms and silent mutations aberrations were detected in IDH1 (1/81; 1.2%), IDH2 (3/81; 3.7%), NPM1 (1/81; 1.2%), and EZH2 (10/81; 12.3%). Another gene recently described in hematological diseases is ASXL1 (additional sex combs like 1) on chromosome 20q11.1. Therefore, the hotspot region of ASXL1 exon 12 was additionally investigated by Sanger sequencing in those 20 cases, in which no mutation had been observed thus far. Nine of these 20 cases (45%) harbored a mutation in ASXL1, thus only 11 cases (13.6%) remained in this cohort in which no mutation was detected. Summarizing this data, 86.4% of these CMMLs harbored at least one molecular aberration with a median of two genes mutated (range 1–4). In more detail, we observed 11 novel distinct EZH2 mutations in ten patients: 7 missense, 3 frameshifts (2 deletions, 1 insertion), and one splice site mutation. EZH2 mutations were found to be heterogeneous and were spread over several exons, predominantly located in the four conserved regions (6/11 in the conserved SET domain; e.g. H680R, N659S). No case revealed a Tyr641 of EZH2 mutation as described for follicular and diffuse large B-cell lymphomas. In median, the burden of EZH2 mutations was 42.5% of sequencing reads per patient (range 1.4–98%). Similarly, a high mutation burden was detected in RUNX1 (median 46.7%), TET2 (median 44.6%), and CBL (median 42.5%) whereas the burden was low in RAS pathway alterations, i.e. NRAS (median 11.1%), KRAS (median 27%), or JAK2 V617F mutations (median 6.9%). With respect to associations of distinct mutations no specific pattern was observed, i.e. EZH2 mutations were concomitantly detected with TET2 (4/10), RUNX1 (3/10), CBL (3/10), JAK2 (3/10), NRAS (2/10), KRAS (1/10), and IDH2 (1/10), respectively. Further, EZH2 mutations were associated neither with morphologic CMML subtype or dysplastic or myeloproliferative characteristics nor with age, white blood cell count, thrombocytes count, or hemoglobin. However, with respect to clinical data a very poor outcome was observed for patients that carried EZH2 mutations compared to EZH2 wild-type cases (median OS 4.3 vs. 130.4 months; p<0.001). In contrast, a significantly better outcome was seen for patients who carried TET2 mutations compared to TET2 wild-type cases (median OS 130.4 vs. 53.6 months, p=0.013). Subsequently, we performed a survival analysis taking both EZH2 and TET2 mutations into account. Here, the cohort was significantly separated into three distinct prognostic groups, i.e. EZH2-mutated with a poor median OS of 4.3 months, EZH2/TET2 wild-type with a median OS of 90 months and TET2-mutated cases with a median OS of 130.4 months (p<0.001). In conclusion, our study revealed molecular mutations in 86.4% of 81 CMML patients providing new insights into the molecular heterogeneity of this disease. Besides alterations in TET2, CBL, ASXL1, and the RAS pathway, EZH2 is targeted by various types of frameshift and point mutations and is a novel biomarker with unfavorable prognosis and clinical utility. Disclosures: Grossmann: MLL Munich Leukemia Laboratory: Employment. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Eder:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership, Research Funding. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

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A Phase I/II Trial of the KSP Inhibitor ARRY-520 In Relapsed/Refractory Multiple Myeloma

Blood ◽

10.1182/blood.v116.21.1959.1959 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1959-1959 ◽

Cited By ~ 4

Author(s):

Jatin J Shah ◽

Jeffrey A. Zonder ◽

Adam Cohen ◽

Donna Weber ◽

Sheeba Thomas ◽

...

Keyword(s):

Multiple Myeloma ◽

Dose Escalation ◽

Research Funding ◽

Phase 1 ◽

Single Agent ◽

Clinical Activity ◽

Employment Equity ◽

Heavily Pretreated ◽

Equity Ownership ◽

Ksp Inhibitor

Abstract Abstract 1959 Background: Kinesin Spindle Protein (KSP) is required for cell cycle progression through mitosis. Inhibition of KSP induces mitotic arrest and cell death. ARRY-520 is a potent, selective KSP inhibitor. Cancers such as multiple myeloma (MM) which depend on the short-lived survival protein MCL-1 are highly sensitive to treatment with ARRY-520. ARRY-520 shows potent activity in preclinical MM models, providing a strong rationale for its clinical investigation in this disease. Methods: This Phase 1 study was designed to evaluate the safety and pharmacokinetics (PK) of ARRY-520 administered intravenously (IV) on Day 1 and Day 2 q 2 weeks without/with granulocyte-colony stimulating factor (G-CSF). Patients (pts) with relapsed/refractory (RR) MM with 2 prior lines of therapy (including both bortezomib and an immunomodulatory agent, unless ineligible for or refusing to receive this therapy) were eligible. Cohorts of at least 3 pts were enrolled in a classical 3 + 3 dose escalation design. Pts were treated for 2 cycles (4 weeks) to evaluate safety prior to dose escalation. Results: Twenty five pts have been treated to date, with a median age of 60 years (range 44–79) and a median of 5 prior regimens (range 2–16). All pts received prior bortezomib or carfilzomib, 21 pts received prior lenalidomide, 17 pts prior thalidomide, and 18 pts had a prior stem cell transplant. Pts received ARRY-520 without G-CSF at 1 mg/m2/day (n = 3), and at 1.25 mg/m2/day (n = 7, 6 evaluable). A dose-limiting toxicity (DLT) of Grade 4 neutropenia was observed at 1.25 mg/m2/day, and this was considered the maximum tolerated dose (MTD) without G-CSF. As neutropenia was the DLT, dose escalation with prophylactic G-CSF support was initiated, at doses of 1.5 mg/m2/day (n = 7, 6 evaluable), 2.0 mg/m2/day (n = 6) and 2.25 mg/m2/day (n = 2) with G-CSF. Both the 2.0 mg/m2/day and 2.25 mg/m2/day dose levels were determined to be non-tolerated, with DLTs of febrile neutropenia (FN) (2 pts at 2.0 mg/m2/day and both pts at 2.25 mg/m2/day) and Grade 3 mucositis (both pts at 2.25 mg/m2/day). One out of 6 evaluable pts at 1.5 mg/m2/day also developed a DLT of FN. In an attempt to optimize the Phase 2 dose, an intermediate dose level of 1.75 mg/m2/day with G-CSF is currently being evaluated. The most commonly reported treatment-related adverse events (AEs) include those observed with other KSP inhibitors, such as hematological AEs (thrombocytopenia, neutropenia, anemia, leukopenia), fatigue, mucositis and other gastro-intestinal AEs. Pts displayed linear PK, a low clearance and a moderate volume of distribution, with moderate-to-high inter-individual variability in PK parameters. The median terminal elimination half life is 65 hours. The preliminary efficacy signal as a single agent is encouraging with 2 partial responses (PR) observed to date per IMWG and EBMT criteria in a heavily pretreated population (23 evaluable pts). A bortezomib-refractory pt with 8 prior lines of therapy, including a tandem transplant, treated at 1 mg/m2/day of ARRY-520 obtained a PR after Cycle 6, with urine protein and kappa light chain levels continuing to decline over time. He remains on-study after 15 months of ARRY-520 treatment. A pt with 2 prior lines of therapy, including prior carfilzomib, has obtained a PR after Cycle 8 at 2 mg/m2/day of ARRY-520, and she is currently ongoing after 4.5 months on therapy. Fifteen pts had a best response of stable disease (SD), including 1 pt with a thus far unconfirmed minimal response, and 6 had progressive disease. A total of 10 pts (43%) achieved a PR or SD lasting > 12 weeks. Several additional pts have shown other evidence of clinical activity, with decrease in paraproteins, increase in hemoglobin levels and regression of plasmacytomas. The median number of cycles is 4 (range 1–28+). Treatment activity has not correlated with any baseline characteristics or disease parameters to date. Conclusions: : The selective KSP inhibitor ARRY-520 has been well tolerated, and shows promising signs of single agent clinical activity in heavily pretreated pts with RR MM. Prophylactic G-CSF has enabled higher doses to be tolerated. No cardiovascular or liver enzyme toxicity has been reported. Enrollment is ongoing at 1.75 mg/m2/day with G-CSF support, and a planned Phase 2 part of the study will be initiated as soon as the MTD is determined. Complete Phase 1 data will be disclosed at the time of the meeting. Disclosures: Shah: Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Millennium: Research Funding. Off Label Use: Revlimid (lenalidomide) in combination with dexamethasone is indicated for the treatment of multiple myeloma patients who have received at least one prior therapy. Zonder:Millennium: Consultancy, Myeloma and Amyloidosis Patient Day Symposium – Corporate support from multiple sponsors for a one-day educational event, Research Funding; Celgene:; Novartis:; Proteolix: . Weber:novartis-unpaid consultant: Consultancy; Merck- unpaid consultant: Consultancy; celgene- none for at least 2 years: Honoraria; millenium-none for 2 years: Honoraria; celgene, Millenium, Merck: Research Funding. Wang:Celgene: Research Funding; Onyx: Research Funding; Millenium: Research Funding; Novartis: Research Funding. Kaufman:Celgene: Consultancy, Honoraria, Research Funding; Millenium: Consultancy, Honoraria; Merck: Research Funding; Genzyme: Consultancy. Walker:Array Biopharma: Employment, Equity Ownership. Freeman:Array Biopharma: Employment, Equity Ownership. Rush:Array Biopharma: Employment, Equity Ownership. Ptaszynski:Array Biopharma: Consultancy. Lonial:Millennium, Celgene, Bristol-Myers Squibb, Novartis, Onyx: Advisory Board, Consultancy; Millennium, Celgene, Novartis, Onyx, Bristol-Myers Squibb: Research Funding.

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A Selective PIM Kinase Inhibitor Is Highly Active In Multiple Myeloma: Mechanism of Action and Signal Transduction Studies

Blood ◽

10.1182/blood.v116.21.4084.4084 ◽

2010 ◽

Vol 116 (21) ◽

pp. 4084-4084 ◽

Cited By ~ 7

Author(s):

Veerendra Munugalavadla ◽

Leanne Berry ◽

Yung-Hsiang Chen ◽

Gauri Deshmukh ◽

Jake Drummond ◽

...

Keyword(s):

Multiple Myeloma ◽

Cell Lines ◽

Combination Treatment ◽

Kinase Inhibitor ◽

Single Agent ◽

Myeloma Cell ◽

Negative Regulator ◽

Combination Drug ◽

Employment Equity ◽

Equity Ownership

Abstract Abstract 4084 Related work from our group has shown the therapeutic utility of PIM inhibition in multiple myeloma cell lines, xenografts, and primary patient samples (Ebens A. et al., ASH 2010 submitted abstr.). In this study we provide detailed mechanistic findings to show that PIM kinase inhibition co-regulates several important elements of the PI3K/AKT/mTOR pathway, resulting in significant synergy for combination drug treatments. The PIM kinases are a family of 3 ser/thr growth factor- & cytokine-induced proteins hypothesized to have redundant survival and growth functions. GNE-652 is a pan-PIM kinase inhibitor with picomolar biochemical potencies and an excellent kinase selectivity profile. Myeloma cell lines exhibit sensitivity to single agent PIM inhibition and a striking synergy in combination with the PI3K inhibitor GDC-0941. Cells respond to this combination with cell cycle arrest and marked apoptosis in vitro. We tested a panel of selective PI3K/AKT/mTOR inhibitors and found PI3K and AKT inhibitors showed the greatest extent of synergy with GNE-652, whereas mTOR inhibitors were synergistic to a lesser extent. These results suggest that PIM signaling converges on both TORC1 and AKT to generate these differential synergies. BAD is a negative regulator of both Bcl-2 and Bcl-XL, and we were able to confirm previous reports that AKT and PIM cooperate to inactivate BAD (Datt et al., 1997; Yan et al., 2003). Pim has been shown to potentially inactivate PRAS40, a negative regulator of TORC1 (Zhang et al., 2009). We demonstrate that PIM or PI3K inhibition caused a loss of phosphorylation on PRAS40 and results in a physical association of PRAS40 and TORC1 and a decrease in phosphorylated p70S6K and S6RP. These reductions were apparent in 7 of 7 cell lines assayed and enhanced by the combination of PI3K and PIM inhibition in these cell lines. Consistent with prior reports (Hammerman et al., 2005), we show that a second node of convergence between PIM and TORC1 is 4E-BP1. Both GDC-0941 and GNE-652 treatments reduced phosphorylation of 4E-BP1 in 7 of 7 myeloma cell lines. Since dephosphorylated 4E-BP1 competes with eIF4G for the mRNA cap binding factor eIF4E, we assayed immunoprecipitates of eIF4E for the presence of eIF4G and 4E-BP1 and observed increased BP1 and decreased 4G. The combination treatment significantly enhanced the loss of 4G relative to either single agent, and importantly, even at 5× the IC50 concentrations for single agents, combination drug treatment achieved greater extent of effect than single agent treatment. Thus PI3K and PIM pathways are redundant at the level of cap-dependent translational initiation mediated by eIF4E. It has been hypothesized a subset of mRNAs are particularly sensitive to inhibition of cap-dependent translation, and that this includes a number of oncogenes such as cyclin D1. We assayed global protein synthesis in MM1.s cells using 35S-methionine and as expected we observed only a modest ≂∼f20% decrease caused by either GNE-652 or GDC-0941 and this decrease was not enhanced by combination treatment. However, we noted across 7 different myeloma cell lines, strong decreases in levels of cyclin D1 that were enhanced by combination treatment. In summary, we have identified several points at which PIM and PI3K/AKT/mTOR converge to provide synergistic apoptosis in multiple myeloma cell lines. These results provide the rationale for further preclinical development of PIM inhibitors and provide the basis for a possible clinical development plan in multiple myeloma. Disclosures: Munugalavadla: Genentech: Employment, Equity Ownership. Berry:Genentech: Employment, Equity Ownership. Chen:Genentech: Employment, Equity Ownership. Deshmukh:Genentech: Employment, Equity Ownership. Drummond:Genentech: Employment, Equity Ownership. Du:Genentech: Employment, Equity Ownership. Eby:Genentech: Employment, Equity Ownership. Fitzgerald:Genentech: Employment, Equity Ownership. S.Friedman:Genentech: Employment, Equity Ownership. E.Gould:Genentech: Employment, Equity Ownership. Kenny:Genentech: Employment, Equity Ownership. Maecker:Genentech: Employment, Equity Ownership. Moffat:Genentech: Employment, Equity Ownership. Moskalenko:Genentech: Employment, Equity Ownership. Pacheco:Genentech: Employment, Equity Ownership. Saadat:Genentech: Employment, Equity Ownership. Slaga:Genentech: Employment, Equity Ownership. Sun:Genentech: Employment, Equity Ownership. Wang:Genentech: Employment, Equity Ownership. Yang:Genentech: Employment, Equity Ownership. Ebens:Genentech Inc: Employment, Equity Ownership.

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