STAG2 Mutations Alter Cohesin Ring Function and Provide Therapeutic Vulnerabilities in Acute Myeloid Leukemia

Abstract Recurrent somatic mutations in core components and modulators of the cohesin ring - a multimeric protein complex that forms a ring structure around DNA and provides spatial genome organization - have been identified across multiple cancer types, including acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS), where they are associated with poor overall survival. Cohesin proteins are involved in sister chromatid cohesion, chromatin organization into loops, transcriptional activation, and DNA damage repair. The mechanisms underlying clonal expansion of these driver mutations are unknown and no therapies have selective efficacy in cohesin-mutant cancers. We sought to determine the effects of mutations in the most frequently mutated cohesin subunit, STAG2, on cohesin complex composition using immunoprecipitation followed by quantitative mass spectrometry (IP-MS), genetic dependencies of STAG2-mutant cells by genome-wide CRISPR screening, and mutant cohesin association with chromatin using chromatin immunoprecipitation followed by sequencing (ChIP-Seq). Our goal was to understand how these mutations contribute to cellular transformation and to identify possible therapeutic targets. Applying IP-MS in AML cell lines engineered with different STAG2 mutations, we identified and validated a switch from STAG2- to its paralog STAG1-containing cohesin complexes. In addition, we observed changes in the interaction of the mutant cohesin complex with proteins involved in DNA repair and replication, including PARP1, and RNA-mediated interaction with RNA splicing machinery, including SF3B family members. We next hypothesized that these cohesin-dependent alterations could lead to shifts in genetic dependencies. Using genome-scale CRISPR-Cas9 screens, we identified preferential dependency of STAG2-mutant cells on STAG1, consistent with our proteomics studies. We also found a striking concordance between additional cellular processes highlighted by IP-MS experiments and observed increased dependency of STAG2-mutant cells on DNA damage repair and mRNA processing. Therefore, STAG2 mutations lead to changes in cohesin complex structure and alter interactions with proteins involved in DNA damage, replication, and RNA modification, which become genetic dependencies in this context. Prompted by this concordance, we evaluated DNA replication, DNA damage and splicing in cohesin-mutant cells. We observed a 4-fold increase in replication fork stalling in STAG2-mutant cells, which was associated with accumulation of double strand DNA breaks and activation of the ATR and ATM DNA damage checkpoints. STAG2-mutant cells demonstrated ~100-fold increased sensitivity to the PARP inhibitor talazoparib, which was consistent across models of other cohesin-mutant subunits. In addition, cohesin-mutant cells showed aberrant splicing and increased sensitivity to treatment with SF3B1 inhibitors E7107 and H3B-8800. In aggregate, genetic or pharmacologic perturbation of DNA damage repair or splicing created a synthetic vulnerability for cohesin-mutant cells in vitro and in vivo. Finally, we explored how STAG1-containing complexes alter cohesin-mediated genome compartmentalization in cohesin-mutant cells. Using ChIP-Seq, we observed that STAG2 loss leads to a global decrease in cohesin binding to chromatin, including at sites of insulated neighborhood boundaries, with subsequent gene expression changes. Loss of cohesin binding was associated with increased enhancer activity and super-enhancer expansion in STAG2-mutant cells. In addition, we identified changes in the co-localization of the mutant cohesin complex with super-enhancer enriched factors, DNA damage repair and splicing machinery. These findings are consistent with a model in which wild type and mutant cohesin complexes, defined by their unique composition and patterns of chromatin binding and architecture, have differential abilities to maintain chromatin organization as it relates to spatial organization of super-enhancers, coactivators and transcription factors, as well as DNA damage repair and splicing machinery. Perturbation of any of these components, which have been recently proposed to form phase-separated nuclear bodies, creates vulnerabilities that may be exploited therapeutically with existing drugs in patients with cohesin-mutated malignancies. Disclosures Abraham: Syros Pharmaceuticals: Equity Ownership. Seiler:H3 Biomedicine: Employment. Buonamici:H3 Biomedicine: Employment. D'Andrea:Intellia Therapeutics: Consultancy; Cedilla Therpeutics: Consultancy, Equity Ownership; EMD-Serono: Consultancy, Research Funding; Sierra: Consultancy, Research Funding; Ideaya: Consultancy, Equity Ownership; Lilly: Consultancy, Research Funding; Formation Biologics: Consultancy. Young:Omega Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Syros Pharmaceuticals: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Camp4 Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Download Full-text

BRCA1/2 Containing Complex 3 (BRCC36) Is Recurrently Mutated in AML with t(8;21) and Associated with Increased Sensitivity to Chemotherapy through Impairment of the DNA Damage Repair Pathway

Blood ◽

10.1182/blood-2018-99-114143 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1487-1487

Author(s):

Tatjana Meyer ◽

Nikolaus Jahn ◽

Anna Dolnik ◽

Peter Paschka ◽

Verena I. Gaidzik ◽

...

Keyword(s):

Dna Damage ◽

Board Of Directors ◽

Cell Lines ◽

Research Funding ◽

De Novo ◽

Dna Damage Repair ◽

Advisory Committees ◽

Data Set ◽

Damage Repair ◽

De Novo Aml

Abstract Introduction BRCA1/BRCA2-containing complex 3 (BRCC36) is a Lys63-specific deubiquitinating enzyme (DUB) involved in DNA damage repair. Mutations in BRCC36 have been identified in 2-3% of patients with myelodysplastic syndromes (MDS) and secondary AML (sAML). The role of BRCC36 mutations in de novo AML and their impact on DNA damage-inducing cytotoxic chemotherapy sensitivity is not clear. Aim We aimed to determine the incidence of BRCC36 mutations in AML and their impact on outcome and drug sensitivity in vitro. Methods We analyzed the entire coding region of BRCC36 for mutations in 191 AML cases with t(8;21) (q22;q22.1) and 95 cases with inv(16) (p13.1q22) using a customized targeted sequencing panel. Data for de novo AML was derived from The Cancer Genome Atlas Research Network (TCGA) data set (NEJM 2013). Lentiviral CRISPR/Cas9 was used to inactivate BRCC36 in t(8;21)-positive AML cell lines - Kasumi-1 and SKNO-1 - and murine hematopoietic stem and progenitor cells (LSKs). Knockout was confirmed by a cleavage assay as well as Western blot. AML1-ETO-9a was expressed by a retroviral vector. Cell lines and LSK cells were treated with different concentrations of doxorubicin or cytarabine and their viability was assessed seven days post treatment. DNA damage was assessed through phospho-γH2AX staining using flow-cytometry. Results BRCC36 mutations were identified in 7 out of 191 patients (3.7%) with t(8;21) AML and none of 95 patients with inv(16). In the TCGA data set one out of 200 patients (0.5%) with de novo AML had a BRCC36 mutation. This patient had a complex karyotype and would be considered as secondary AML with myelodysplastic-associated changes according to the 2016 WHO classification. Six of the 7 mutations were missense or nonsense mutations that were predicted to be deleterious to BRCC36 function. One mutation affected a splice site at exon 6, resulting in an impaired splicing capability. With intensive standard chemotherapy all patients with BRCC36 mutations achieved a complete remission and had an estimated relapse-free and overall survival of 100% after a median follow up of 4.2 years. Given its role in DNA damage repair, we hypothesized that BRCC36 inactivation sensitizes AML cells to DNA-damage inducing drugs. In order to test this, we generated BRCC36 knockout Kasumi-1 and SKNO-1 cell lines using CRISPR-Cas9. BRCC36 inactivation had no impact on cell growth on either of the cell lines. However, we found that BRCC36 knockout cells were significantly more sensitive to doxorubicin as compared to the parental cells with normal BRCC36. This was accompanied by a significant increase in DNA damage as assessed by phospho-γH2AX in BRCC36 knockout vs control cells after doxorubicin treatment. In contrast, BRCC36 inactivation had no impact on cytarabine sensitivity. We next assessed drug sensitivity in primary murine leukemic cells expressing AML1-ETO-9a. Again, inactivation of BRCC36 resulted in a significant higher sensitivity to doxorubicin but not cytarabine. Conclusion We found BRCC36 to be recurrently mutated in t(8;21)-positive AML Inactivation of BRCC36 was associated with impairment of the DNA damage repair pathway and thus higher sensitivity to DNA damage-inducing chemotherapy. This might be also reflected by the favorable clinical outcome of patients with BRCC36 mutated t(8;21)-positive AML, a finding which has to be confirmed in a large patient cohort. Disclosures Paschka: Pfizer: Membership on an entity's Board of Directors or advisory committees; Takeda: Other: Travel support; Novartis: Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Speakers Bureau; Otsuka: Membership on an entity's Board of Directors or advisory committees; Sunesis: Membership on an entity's Board of Directors or advisory committees; Jazz: Speakers Bureau; Amgen: Other: Travel support; Janssen: Other: Travel support; Bristol-Meyers Squibb: Other: Travel support, Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Speakers Bureau; Astellas: Membership on an entity's Board of Directors or advisory committees, Travel support; Astex: Membership on an entity's Board of Directors or advisory committees; Agios: Membership on an entity's Board of Directors or advisory committees. Bullinger:Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pfizer: Speakers Bureau; Bayer Oncology: Research Funding; Sanofi: Research Funding, Speakers Bureau; Janssen: Speakers Bureau; Bristol-Myers Squibb: Speakers Bureau; Amgen: Honoraria, Speakers Bureau; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Döhner:Novartis: Consultancy, Honoraria, Research Funding; Jazz: Consultancy, Honoraria; Jazz: Consultancy, Honoraria; AROG Pharmaceuticals: Research Funding; Janssen: Consultancy, Honoraria; Celator: Consultancy, Honoraria; Pfizer: Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Astex Pharmaceuticals: Consultancy, Honoraria; AROG Pharmaceuticals: Research Funding; Janssen: Consultancy, Honoraria; Seattle Genetics: Consultancy, Honoraria; Sunesis: Consultancy, Honoraria, Research Funding; Astellas: Consultancy, Honoraria; Astex Pharmaceuticals: Consultancy, Honoraria; Bristol Myers Squibb: Research Funding; Pfizer: Research Funding; Agios: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Agios: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; Celator: Consultancy, Honoraria; Astellas: Consultancy, Honoraria; Bristol Myers Squibb: Research Funding; Seattle Genetics: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Sunesis: Consultancy, Honoraria, Research Funding.

Download Full-text

Therapy-Related Myeloid Neoplasm Has a Distinct Pro-Inflammatory Bone Marrow Microenvironment and Delayed DNA Damage Repair

Blood ◽

10.1182/blood-2020-137277 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 37-38

Author(s):

Monika M Kutyna ◽

Li Yan A Wee ◽

Sharon Paton ◽

Dimitrios Cakouros ◽

Agnieszka Arthur ◽

...

Keyword(s):

Dna Damage ◽

Board Of Directors ◽

Research Funding ◽

High Dose ◽

Cytotoxic Therapy ◽

Advisory Committees ◽

Myeloid Neoplasm ◽

Myeloid Neoplasms ◽

Damage Repair

Introduction: Therapy-related myeloid neoplasms (t-MN) are associated with extremely poor clinical outcomes in otherwise long-term cancer survivors. t-MN accounts for ~20% of cases of myeloid neoplasms and is expected to rise due to the increased use of chemotherapy/radiotherapy (CT/RT) and improved cancer survivorship. Historically, t-MN was considered a direct consequence of DNA damage induced in normal hematopoietic stem cells (HSC) by DNA damaging cytotoxics. However, these studies have largely ignored the bone marrow (BM) microenvironment and the effects of age and concurrent/previous cancers. Aim: We performed an exhaustive functional study of mesenchymal stromal cells (MSC) obtained from a comparatively large cohort of t-MN patients and carefully selected control populations to evaluate the long-term damage induced by cytotoxic therapy to BM microenvironment and its impact on malignant and normal haematopoiesis. Methods: Four different cohorts were used: (1) t-MN, in which myeloid malignancy occurred after CT/RT for a previous cancer (n=18); (2) patients with multiple cancer and in which a myeloid neoplasm developed following an independent cancer which was not treated with CT/RT (MC-MN; n=10); (3) primary MN (p-MN; n=7) untreated and without any prior cancer or CT/RT; (4) age-matched controls (HC; n=17). Morphology, proliferation, cellular senescence, differentiation potential and γH2AX DNA damage response was performed. Stem/progenitor supportive capacity was assessed by co-culturing haematopoietic stem cells on MSC feeder-layer in long-term culture initiating assay (LTC-IC). Cytokine measurements were performed using 38-plex magnetic bead panel (Millipore) and RNA sequencing libraries were prepared with Illumina TruSeq Total RNA protocol for 150bp paired-end sequencing on a NextSeq500 instrument. Functional enrichment analysis was performed using EnrichR software. Results: MSC cultured from t-MN patients were significantly different from HC, p-MN and MC-MN MSC according to multiple parameters. They exhibited aberrant morphology consisting of large, rounded and less adhesive cells compared to typical spindle-shaped morphology observed with controls. MSC from myeloid neoplasm also showed impaired proliferation, senescence, osteo- and adipogenic differentiation with t-MN MSC showing the greatest differences. DNA repair was dramatically impaired compared to p-MN and HC (Fig.1A). Importantly, these aberrant t-MN MSC were not able to support normal or autologous in vitro long-term haematopoiesis (Fig.1B). The biological characteristic and poor haematopoietic supportive capacity of MSC could be "cell-intrinsic" or driven by an altered paracrine inflammatory microenvironment. Interestingly, several inflammatory cytokines were higher in t-MN compared with marrow interstitial fluid obtained from p-MN patients (Fig.1Ci) and many of these including Fractalkine, IFNα2, IL-7 and G-CSF were also significantly higher in t-MN MSC conditional media (Fig.1Cii). Together, this data suggest that t-MN microenvironment is distinct from p-MN with paracrine production of pro-inflammatory milieu that may contribute to poor HSC supportive capacity. Preliminary whole transcriptome analysis revealed differential gene expression between t-MN and HC (Fig.1Di) and p-MN MSC. Importantly, the deregulated genes play critical role in cell cycle, DNA damage repair, and cellular senescence pathways explaining phenotypical characteristic of t-MN MSC (Fig.1Dii). Moreover CXCL12 expression, a key regulator of haematopoiesis, was significantly lower in t-MN compared to HC (p=0.002) and p-MN MSC (p=0.009), thus explaining poor HSC supportive capacity. The key difference between the p-MN, MC-MN and t-MN is prior exposure to CT/RT. To study this we obtained MSC from two t-MN patients for whom we had samples at the time of their primary cancer, post high-dose chemotherapy and at the time of t-MN. MSC displayed aberrant proliferation and differentiation capacity after high-dose cytotoxic therapy (2 to 4 years prior to developing t-MN) and remained aberrant at t-MN diagnosis (Fig.1E). Conclusions: BM-MSC from t-MN patients are significantly abnormal compared with age-matched controls and typical myeloid neoplasm. Importantly, prior CT/RT leads to long-term irreversible damage to the BM microenvironment which potentially contributes to t-MN pathogenesis. Disclosures Hughes: Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Hiwase:Novartis Australia: Research Funding.

Download Full-text

Targeting Replicative Stress to Treat Hematological Disorders

Blood ◽

10.1182/blood.v126.23.2419.2419 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2419-2419

Author(s):

Francesca Cottini ◽

Giovanni Tonon ◽

Teru Hideshima ◽

Paul G. Richardson ◽

Kenneth C Anderson

Keyword(s):

Dna Damage ◽

Board Of Directors ◽

Cell Lines ◽

Research Funding ◽

Hematological Malignancies ◽

Advisory Committees ◽

Hematological Disorders ◽

Replicative Stress ◽

Hematological Cancers ◽

Equity Ownership

Abstract Background: Combinations of chromosomal translocations, copy-number variations, somatic mutations, and clonal heterogeneity that characterize hematological cancers make every patient unique from a genetic point of view. This variety creates a true challenge for tailored therapy. We have previously described that myeloma (MM) cells present signs of ongoing DNA damage, and activate an ATM/ABL1-dependent DNA damage response (DDR) without overt apoptosis. Here we further characterize the mechanisms of DNA damage and replicative stress in MM, and we extend this knowledge to other hematological malignancies to evaluate a novel and possibly shared approach to synthetic lethality (1). Results: We studied a panel of MM cell lines together with acute myeloid, lymphoid leukemia and lymphoma cell lines. Several cell lines have demonstrable ongoing DNA damage, activate ATR and CHK1 and also present with signs of replicative stress, such as 53BP1, RPA and RAD51 foci. We next evaluated a gene expression signature specific for increased chromosomal instability and DNA damage in a cohort of MM patients, comparing them with normal plasma cells. Specifically, we identified a subset of patients, representing around 20 percent of individuals with MM that show this signature and also present with an unfavorable prognosis due to a more aggressive disease. Interestingly, in a multivariate analysis, this signature was independent from other poor prognostic criteria, including proliferation and the presence of MMSET/FGFR3 or MAF translocations (2), hence representing a potential novel prognostic signature (3). Gene-set enrichment studies are ongoing in other hematological disorders. However, preliminary data show that subsets of patients with other hematological malignancies also present with intense over expression of genes belonging to the instability signature when compared to normal B cells, in a very similar fashion to that seen with MM. An intact ATR/CHK1 pathway is crucial for the survival of tumor cells in vivo, especially in the presence of activated oncogenes. For instance, Em-myc transgenic mice develop B-cell lymphomas with intense replicative stress that can be blocked by crossing the Em-myc transgenic mice with a hypomorphic Atr mouse strain (Atr-Seckel; Atr S/S). We therefore decided to exploit the concept of replicative stress overload, impeding the capacity of the cells for repairing the excess of damaged DNA. We then silenced ATR using shRNAs, the upstream protein involved in the control of stalled replication origins, in two MM cell lines (H929 and OPM-2) and in the Jurkat cell line. Critically, inhibition of ATR caused a reduction in cellular growth and induction of apoptosis. A similar phenotype was observed using VE-821, a specific ATR inhibitor. Finally, a broad panel of MM cells and leukemia cell lines was used to confirm these growth inhibitory effects. Conclusion: Replicative stress is present in multiple groups of patients with aggressive types of MM or leukemia. Strategies which couple pre-existing high rates of DNA damage with reduced DNA repair can specifically cause apoptosis of malignant cells and encouragingly spare normal ones, thus providing a strong rationale for potential clinical benefit to those cohorts of patients with otherwise very unfavorable outcomes. (1) Cottini F, Hideshima T, Xu C, Sattler M, Dori M, Agnelli L, et al. Rescue of Hippo coactivator YAP1 triggers DNA damage-induced apoptosis in hematological cancers. Nature medicine. 2014;20(6):599-606. (2) Zhan F, Huang Y, Colla S, Stewart JP, Hanamura I, Gupta S, et al. The molecular classification of multiple myeloma. Blood. 2006;108(6):2020-8. (3) Cottini F, Teru Hideshima, Rikio Suzuki, Yu-Tzu Tai, et al. Synthetic lethal approaches exploiting DNA damage in aggressive myeloma. Cancer Discovery ahead of print. Disclosures Richardson: Novartis: Membership on an entity's Board of Directors or advisory committees; Millennium Takeda: Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Gentium S.p.A.: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees. Anderson:Celgene Corporation: Consultancy; Oncocorp: Equity Ownership; acetylon pharmaceuticals: Equity Ownership; Gilead: Consultancy; BMS: Consultancy; Millennium: Consultancy.

Download Full-text

Single-Cell Mapping of Stress Response and Cell Death Pathways in Acute Myeloid Leukemia Reveals Stressor-Specific Alterations and Distinct Response Patterns

Blood ◽

10.1182/blood-2019-131784 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 882-882

Author(s):

Muharrem Muftuoglu ◽

Vivian Ruvolo ◽

Yuki Nishida ◽

Po Yee Mak ◽

Peter P. Ruvolo ◽

...

Keyword(s):

Dna Damage ◽

Cell Death ◽

Stress Response ◽

Er Stress ◽

Single Cell ◽

Board Of Directors ◽

Research Funding ◽

Cell Mapping ◽

Advisory Committees ◽

Equity Ownership

Background: Cells respond to stress in various ways ranging from adaptation to environmental challenges and activation of survival pathways to induction of cell death. The initial response to stress encompasses adaptive measures to ensure survival and in the presence of irreparable damage associated with unresolved stress cell death ensues. Understanding the principles and mechanisms governing cell survival over cell death is of particular importance in the field of cancer therapy. It is intriguing that exposure of a seemingly homogenous population to death inducing stimuli, such as chemotherapeutic agents, induces fractional tumor killing in a stochastic manner while a subgroup of cells acquire a persistent state, most probably through activation of compensatory survival pathways. Fractional cell killing and, therefore, inability to completely eradicate transformed cells result in resistance to therapy. Methods/Results: To gain further insight into compensatory mechanisms and divergent responses elicited in response to death inducing stimuli we designed a multiparametric flow cytometry panel for simultaneous assessment of different forms of cell death at the single cell level, and aimed to dissect stimulus-specific death patterns and pinpoint potential compensatory mechanisms in persistent cells. We modified ( Bergamaschi et al. 2019) and utilized panels including antibodies against RIP3, LC3B, cleaved caspase 3, cleaved PARP-1, PERK, H2AX, p21, Ki-67 and dead cell discriminating dye. This enabled simultaneous interrogation of a multitude of cell death modes including necrosis, necroptosis, apoptosis and parthanatos in response to DNA damage and as well as proliferation, autophagy and endoplasmic reticulum (ER) stress. To test this concept, we initially utilized agents inducing DNA damage and generated two-dimensional t-SNE plots and diffusion maps to illustrate the multifaceted stress response and developmental trajectories upon challenging with DNA damaging agents. Exposure of acute myeloid leukemia (AML) cell lines to etoposide (E) and daunorubicin (DNR) dramatically altered cellular landscape and resulted in emergence of distinct stress responses characterized by differential induction of autophagy, ER stress and DNA damage response and an increase in multiple cell death subpopulations differentially expressing cleaved caspase 3, PARP-1, necrotic cell identifier (live dead aqua dye) and H2AX. We then generated diffusion maps to infer developmental trajectories of dead cells and identified H2AX+PARP+Caspase-3 co-expression as the earliest event occurring in dying cells while cells stained positive for dead cell dye only marked the latest stage. Of note, a fraction of cells exhibited increased autophagy, accompanied with high ER stress and low DNA damage. Presumably, this pattern identifies persistent cells attaining a transient state in response to E and DNR associated with higher likelihood of survival. Evidently, external stress induced a divergent multifaceted response: DNA damage followed by cell death vs. induction of adaptive mechanisms including autophagy and high ER stress. Although both E and DNR preferentially targeted proliferating cells and induced cell cycle arrest, overall stress response to E was distinct from stress to DNR in high-dimensional plane. To attain a comprehensive overview of stress response to E vs. DNR we compared t-SNE maps depicting overall stress response and observed significant segregation. Autophagy and ER stress was more pronounced in E group while DNR completely abrogated proliferation in surviving cells. To further corroborate the utility of this approach, we assessed the activity of exportin-1 (XPO1, KPT-330) and MDM2 (DS-3032b) inhibitors. KPT-330 and DS-3032b individually induced limited cell death. Combination of XPO-1 and MDM2 inhibitors resulted in enhanced apoptotic cell death with unrepaired DNA damage while surviving cells displayed an autophagy pattern. Conclusion: These findings provide proof of concept for the utility of single cell mapping of cellular stress in delineating stressor-specific response patterns and identifying potential resistance mechanisms. Single cell mapping of cell stress and cell death can inform the development of more effective combinatorial drug regimens. Studies to identify stress signatures of targeted agents currently developed for the treatment of AML are ongoing Figure 1 Disclosures Carter: Amgen: Research Funding; AstraZeneca: Research Funding; Ascentage: Research Funding. Andreeff:NCI-RDCRN (Rare Disease Cliln Network): Membership on an entity's Board of Directors or advisory committees; CLL Foundation: Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy; AstaZeneca: Consultancy; 6 Dimensions Capital: Consultancy; German Research Council: Membership on an entity's Board of Directors or advisory committees; Leukemia Lymphoma Society: Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Consultancy; Daiichi Sankyo, Inc.: Consultancy, Patents & Royalties: Patents licensed, royalty bearing, Research Funding; BiolineRx: Membership on an entity's Board of Directors or advisory committees; NCI-CTEP: Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy; Center for Drug Research & Development: Membership on an entity's Board of Directors or advisory committees; Cancer UK: Membership on an entity's Board of Directors or advisory committees; NIH/NCI: Research Funding; Breast Cancer Research Foundation: Research Funding; CPRIT: Research Funding; Eutropics: Equity Ownership; Senti Bio: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Oncoceutics: Equity Ownership; Oncolyze: Equity Ownership; Reata: Equity Ownership; Aptose: Equity Ownership.

Download Full-text

Using HSP90 Inhibitors to Target DNA Repair Proteins in AML

Blood ◽

10.1182/blood-2018-99-116485 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 5144-5144

Author(s):

Janani Ravikrishnan ◽

Tzung-Huei Lai ◽

Shaneice Renee Mitchell ◽

Pei-Jung Wu ◽

Shelley Orwick ◽

...

Keyword(s):

Dna Damage ◽

Dna Repair ◽

Mouse Model ◽

Board Of Directors ◽

Dna Damage Repair ◽

Nucleoside Analog ◽

Proper Function ◽

Advisory Committees ◽

Damage Repair ◽

Target Dna

Abstract Acute myeloid leukemia (AML) is a disease of proliferative and abnormally differentiated myeloid cells(Döhner, Weisdorf, & Bloomfield, 2015) that is characterized by chromosomal aberrations and gene mutations that influence outcome(De Kouchkovsky & Abdul-Hay, 2016). Despite the approval of targeted therapies, induction therapy with cytarabine (Ara-C) and anthracycline has remained the standard of care for AML(Dombret & Gardin, 2016; Showel & Levis, 2014). Newer chemotherapeutics such as Sapacitabine have shown comparable clinical outcomes to Ara-C in older patients(Burnett et al., 2015). Ara-C is a nucleoside analog which incorporates during DNA replication and stalls replication(Liu et al., 2010) while Sapacitabine induces single stranded nicks in the DNA leading to double stranded breaks which are repaired by Homologous Recombination (HR)(Burnett et al., 2015; Lim & Jamieson, 2014). However, eventually patients develop resistance to chemotherapy leading to poor prognosis after relapse(Wu, Duan, Chen, & Chen, 2017). One aspect of this is due to upregulation of DNA damage repair proteins leading to increased repair of any DNA lesions primarily through HR. We therefore are interested in developing a way to target DNA repair to extend nucleoside analog therapy in AML. HSP90 is a molecular chaperone that is critical for the folding and proper function of multiple key proteins of various DNA repair pathways(Dote, Burgan, Camphausen, & Tofilon, 2006). Relevant to such repair, ATM and Chk1 become recruited to DNA double strand breaks to activate defined downstream signaling cascades that result in cell cycle arrest and DNA repair(Ceccaldi, Rondinelli, & D'Andrea, 2016). Of these,γ-H2AX acts a sensor of DNA damage and repair by forming foci at the sites of damaged DNA that get resolved upon successful repair(Sharma, Singh, & Almasan, 2012). Similarly, the Rad51 helicase plays a key role in HR repair and the presence of Rad51 foci can be used as a marker for successful HR(Gachechiladze, Škarda, Soltermann, & Joerger, 2017). To determine whether inhibiting HSP90 compromised DNA repair, we treated both AML cell lines OCI-AML3 and MV4-11 with HSP90 inhibitor, onalespib. OCI-AML3 cells show a time dependent decrease in DNA damage repair proteins Chk1 and Rad51 by western blotting. Furthermore, primary patient samples treated with onalespib for 24 and 36 hours also show a depletion of Chk1 and Rad51. We then used ionizing radiation (IR) to induce dsbreaks and induce signaling and repair(Ceccaldi et al., 2016). Using immunofluorescence, we found that in OCI-AML3 after exposure to IR there was a significant increase in number of Rad51 foci after 6 hours. The large number of γ-H2AX foci formed at .5 hour after IR decrease by 6 hours post-IR. This suggests that there is a successful HR repair occurring in this AML cell line. When OCI-AML3 cells were pretreated with onalespib, Rad51 foci are no longer present and γ-H2AX foci remained present at 6 hours suggesting unrepaired dsbreaks. Using the colony forming assay we see that there is a slight decrease in viability of OCI-AML3 cells exposed to onalespib however, in MV4-11 cells there is a steep decline which could be due to the differential expression of genes such as FLT3-ITD, a client protein of HSP90. Low doses of cytarabine or sapacitabine modestly decreased colony growth of OCI-AML3 and MV4-11 cells but exposing cells to onalespib for 24 hours sensitized the cells to both cytarabine and sapacitabine. Finally, we have also evaluated effects of onalespib in an in-vivo MOLM13 driven mouse model of AML and have shown that treatment with onalespib extended survival in this mouse model. Disclosures Mims: Abbvie Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Agios Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy.

Download Full-text

Distinct Senescent Bone Marrow Microenvironment in Therapy-Related Myeloid Neoplasms

Blood ◽

10.1182/blood-2021-148215 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 2585-2585

Author(s):

Monika M Kutyna ◽

Chung Hoow Kok ◽

Sharon Paton ◽

Dimitrios Cakouros ◽

Agnieszka Arthur ◽

...

Keyword(s):

Dna Damage ◽

Board Of Directors ◽

Transcriptome Analysis ◽

Research Funding ◽

Differentially Expressed ◽

Advisory Committees ◽

Irreversible Damage ◽

Myeloid Neoplasms ◽

Damage Repair

Abstract Background: Therapy-related myeloid neoplasms (tMN) is a second haematological malignancy associated with distinct molecular profile (Singhal et al Leukemia 2019) and dismal outcome. tMN is believed to arise from cytotoxic DNA damage to haematopoietic stem cells (HSC). Although, cytotoxic therapy (CT) can damage bone marrow (BM) microenvironment, its role in tMN pathogenesis remains unknown. Aim and Methods: We performed comprehensive multiomic profiling (transcriptomic, cytokine quantification, phenotype, DNA damage) of BM stromal cells (BMSC) from (i) tMN patients previously exposed to CT. Critically, we compared it with (ii) patients with MN and a history of another cancer without CT (pMN+Ca), (iii) primary MN (pMN) and (iv) age-matched controls (Healthy). Results: To decipher microenvironmental changes induced by CT from that of MN and age-related changes, whole transcriptome analysis was performed on BMSC isolated from tMN and compared with all control cohorts. Twenty-nine genes were differentially expressed in tMN compared to Healthy (FDR < 0.1, P < 0.05). Unexpectedly 146 genes were differentially expressed in tMN BMSC compared to other MN and interestingly, ~90% of differentially expressed genes were involved in senescence. Moreover, functional enrichment and GO analysis suggest DNA damage repair, cell cycle regulation, and senescence pathways were deregulated in tMN BMSC (Fig. 1A). Genes such as CDKN1A (a critical cyclin dependent kinase inhibitor orchestrating cell cycle arrest), TNFRSF10D (senescence associated), and FGF-2 (a key player in cell proliferation) were highly expressed (P < 0.001). These findings were validated by demonstrating other features of senescence in tMN BMSC: (i) enlarged/flattened cellular morphology, (ii) decreased cell proliferation and colony-forming potential, (iii) increased β-galactosidase expression, and (iv) defective DNA damage repair (Fig. 1Bi-iv). Interestingly, within the tMN cohort there was no correlation between latency period (the interval between completion of CT until tMN diagnosis) and senescence, indicating that higher senescence is persistent even after several years of CT. Senescence associated secretory phenotype (SASP), a mixture of inflammatory cytokines and chemokines such as IL-7, IL-1β, IL-13, and IL-6, were significantly higher in conditioned media of tMN BMSC (9/14, 64%) (Fig. 1 Bv). Despite reduced proliferation and senescence, transcriptome analysis showed enrichment of metabolic and energy production pathways in tMN BMSC compared to controls. TXNRD1, regulator of glucose and lipid metabolism, and BNIP3, a negative regulator of mitochondrial potential, were highly expressed in tMN BMSC (P < 0.001). These findings were further verified by Seahorse bioenergetic analyses. The overall energetic rate (as assessed by ATP production) was higher in tMN compared to Healthy BMSC (P = 0.002), with higher proportion of ATP generated by glycolysis (77% versus 35.5%) (Fig. 1C). Adipogenic differentiation potential of senescent BMSC is not well known. Transcriptome analysis showed reduced expression of genes involved in adipogenesis in tMN BMSC. This was further validated by two independent in vitro assays showing reduced adipogenesis (Fig. 1D). Interestingly, PNPLA2, a catalyst of the first lipolysis reaction, were significantly de-regulated in tMN BMSC (P < 0.001). The key difference in tMN and other MN is prior exposure to CT. Hence, we hypothesise that prior CT leads to long-term irreversible damage to BM microenvironment and induced senescence, which in turn propagate senescence in surrounding normal cells and promote clonal abnormalities in HSC. Other possibility is that tMN clone can induce these changes in BM microenvironment. To decipher it, we assessed serial BMSC. We observed aberrant stroma proliferation and bi-differentiation capacity, following CT, well before the diagnosis of tMN. Conclusions: By multiple orthogonal indices, our results show that tMN BMSC lie on an extreme trajectory away from normal and typical MN, with massive defect in senescence and distinct metabolic phenotype. Importantly, prior CT leads to long-term irreversible damage to the BM microenvironment which potentially contributes to tMN pathogenesis. Together, these data provide a valuable resource for future strategies to delay or prevent the onset of tMN and assist in marrow regeneration in patients undergoing CT. Figure 1 Figure 1. Disclosures Hughes: BMS: Research Funding; Novartis: Honoraria, Research Funding; Takeda: Honoraria. Hiwase: Novartis: Membership on an entity's Board of Directors or advisory committees; AbbVie: Membership on an entity's Board of Directors or advisory committees.

Download Full-text

Rapid and Robust Mobilization of CD34+ HSCs without G-CSF Following Administration of Mgta-145 Alone or in Combination with Plerixafor

Blood ◽

10.1182/blood-2019-127523 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1961-1961

Author(s):

John F. DiPersio ◽

Jonathan Hoggatt ◽

Steven Devine ◽

Lukasz Biernat ◽

Haley Howell ◽

...

Keyword(s):

Single Dose ◽

Adverse Events ◽

Board Of Directors ◽

Preliminary Data ◽

Research Funding ◽

Fold Change ◽

Employment Equity ◽

Advisory Committees ◽

Cd34 Cells ◽

Equity Ownership

Background Granulocyte colony-stimulating factor (G-CSF) is the standard of care for mobilization of hematopoietic stem cells (HSCs). G-CSF requires 4-7 days of injections and often multiple aphereses to acquire sufficient CD34+ cells for transplant. The number of CD34+ HSCs mobilized can be variable and patients who fail to mobilize enough CD34+ cells are treated with the combination of G-CSF plus plerixafor. G-CSF use is associated with bone pain, nausea, headaches, fatigue, rare episodes of splenic rupture, and is contraindicated for patients with autoimmune and sickle cell disease. MGTA-145 (GroβT) is a CXCR2 agonist. MGTA-145, in combination with plerixafor, a CXCR4 inhibitor, has the potential to rapidly and reliably mobilize robust numbers of HSCs with a single dose and same-day apheresis for transplant that is free from G-CSF. MGTA-145 plus plerixafor work synergistically to rapidly mobilize HSCs in both mice and non-human primates (Hoggatt, Cell 2018; Goncalves, Blood 2018). Based on these data, Magenta initiated a Phase 1 dose-escalating study to evaluate the safety, PK and PD of MGTA-145 as a single agent and in combination with plerixafor. Methods This study consists of four parts. In Part A, healthy volunteers were dosed with MGTA-145 (0.0075 - 0.3 mg/kg) or placebo. In Part B, MGTA-145 dose levels from Part A were selected for use in combination with a clinically approved dose of plerixafor. In Part C, a single dose MGTA-145 plus plerixafor will be administered on day 1 and day 2. In Part D, MGTA-145 plus plerixafor will be administered followed by apheresis. Results MGTA-145 monotherapy was well tolerated in all subjects dosed (Table 1) with no significant adverse events. Some subjects experienced mild (Grade 1) transient lower back pain that dissipated within minutes. In the ongoing study, the combination of MGTA-145 with plerixafor was well tolerated, with some donors experiencing Grade 1 and 2 gastrointestinal adverse events commonly observed with plerixafor alone. Pharmacokinetic (PK) exposure and maximum plasma concentrations increased dose proportionally and were not affected by plerixafor (Fig 1A). Monotherapy of MGTA-145 resulted in an immediate increase in neutrophils (Fig 1B) and release of plasma MMP-9 (Fig 1C). Neutrophil mobilization plateaued within 1-hour post MGTA-145 at doses greater than 0.03 mg/kg. This plateau was followed by a rebound of neutrophil mobilization which correlated with re-expression of CXCR2 and presence of MGTA-145 at pharmacologically active levels. Markers of neutrophil activation were relatively unchanged (<2-fold vs baseline). A rapid and statistically significant increase in CD34+ cells occurred @ 0.03 and 0.075 mg/kg of MGTA-145 (p < 0.01) relative to placebo with peak mobilization (Fig 1D) 30 minutes post MGTA-145 (7-fold above baseline @ 0.03 mg/kg). To date, the combination of MGTA-145 plus plerixafor mobilized >20/µl CD34s in 92% (11/12) subjects compared to 50% (2/4) subjects receiving plerixafor alone. Preliminary data show that there was a significant increase in fold change relative to baseline in CD34+ cells (27x vs 13x) and phenotypic CD34+CD90+CD45RA- HSCs (38x vs 22x) mobilized by MGTA-145 with plerixafor. Mobilized CD34+ cells were detectable at 15 minutes with peak mobilization shifted 2 - 4 hours earlier for the combination vs plerixafor alone (4 - 6h vs 8 - 12h). Detailed results of single dose administration of MGTA-145 and plerixafor given on one day as well as also on two sequential days will be presented along with fully characterized graft analysis post apheresis from subjects given MGTA-145 and plerixafor. Conclusions MGTA-145 is safe and well tolerated, as a monotherapy and in combination with plerixafor and induced rapid and robust mobilization of significant numbers of HSCs with a single dose in all subjects to date. Kinetics of CD34+ cell mobilization for the combination was immediate (4x increase vs no change for plerixafor alone @ 15 min) suggesting the mechanism of action of MGTA-145 plus plerixafor is different from plerixafor alone. Preliminary data demonstrate that MGTA-145 when combined with plerixafor results in a significant increase in CD34+ fold change relative to plerixafor alone. Magenta Therapeutics intends to develop MGTA-145 as a first line mobilization product for blood cancers, autoimmune and genetic diseases and plans a Phase 2 study in multiple myeloma and non-Hodgkin lymphoma in 2020. Disclosures DiPersio: Magenta Therapeutics: Equity Ownership; NeoImmune Tech: Research Funding; Cellworks Group, Inc.: Membership on an entity's Board of Directors or advisory committees; Karyopharm Therapeutics: Consultancy; Incyte: Consultancy, Research Funding; RiverVest Venture Partners Arch Oncology: Consultancy, Membership on an entity's Board of Directors or advisory committees; WUGEN: Equity Ownership, Patents & Royalties, Research Funding; Macrogenics: Research Funding, Speakers Bureau; Bioline Rx: Research Funding, Speakers Bureau; Celgene: Consultancy; Amphivena Therapeutics: Consultancy, Research Funding. Hoggatt:Magenta Therapeutics: Consultancy, Equity Ownership, Research Funding. Devine:Kiadis Pharma: Other: Protocol development (via institution); Bristol Myers: Other: Grant for monitoring support & travel support; Magenta Therapeutics: Other: Travel support for advisory board; My employer (National Marrow Donor Program) has equity interest in Magenta. Biernat:Medpace, Inc.: Employment. Howell:Magenta Therapeutics: Employment, Equity Ownership. Schmelmer:Magenta Therapeutics: Employment, Equity Ownership. Neale:Magenta Therapeutics: Employment, Equity Ownership. Boitano:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Cooke:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Goncalves:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Raffel:Magenta Therapeutics: Employment, Equity Ownership. Falahee:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Morrow:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Davis:Magenta Therapeutics: Employment, Equity Ownership.

Download Full-text

Treatment Patterns and Outcomes in Elderly Patients with Newly Diagnosed Multiple Myeloma: Results from the Connect® MM Registry

Blood ◽

10.1182/blood-2019-126205 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3129-3129

Author(s):

Hans C. Lee ◽

Sikander Ailawadhi ◽

Cristina Gasparetto ◽

Sundar Jagannath ◽

Robert M. Rifkin ◽

...

Keyword(s):

Board Of Directors ◽

Research Funding ◽

Causes Of Death ◽

Age Groups ◽

Treatment Patterns ◽

Age Group ◽

Newly Diagnosed ◽

Employment Equity ◽

Advisory Committees ◽

Equity Ownership

Background: Multiple myeloma (MM) is common among the elderly, with 35% of patients (pts) diagnosed being aged ≥75 years (y). With increasing overall life expectancy, the incidence and prevalence of newly diagnosed and previously treated MM patients ≥80 y is expected to increase over time. Because elderly pts are often excluded from clinical trials, data focused on their treatment patterns and clinical outcomes are lacking. The Connect® MM Registry (NCT01081028) is a large, US, multicenter, prospective observational cohort study of pts with newly diagnosed MM (NDMM) designed to examine real-world diagnostic patterns, treatment patterns, clinical outcomes, and health-related quality of life patient-reported outcomes. This analysis reviews treatment patterns and outcomes in elderly pts from the Connect MM Registry. Methods: Pts enrolled in the Connect MM registry at 250 community, academic, and government sites were included in this analysis. Eligible pts were adults aged ≥18 y with symptomatic MM diagnosed ≤2 months before enrollment, as defined by International Myeloma Working Group criteria; no exclusion criteria were applied. For this analysis, pts were categorized into 4 age groups: <65, 65 to 74, 75 to 84, and ≥85 y. Pts were followed from time of enrollment to the earliest of disease progression (or death), loss to follow-up, or data cutoff date of February 7, 2019. Descriptive statistics were used for baseline characteristics and treatment regimens. Survival outcomes were analyzed using Cox regression. Time to progression (TTP) analysis excluded causes of death not related to MM. Results: Of 3011 pts enrolled (median age 67 y), 132 (4%) were aged ≥85 y, and 615 (20%) were aged 75-84 y at baseline. More pts aged ≥85 y had poor prognostic factors such as ISS stage III disease and reduced hemoglobin (<10 g/dL or >2 g/dL <LLN) compared with other age groups, although no notable differences between creatinine and calcium levels were observed across age groups (Table). A lower proportion of elderly pts (75-84 and ≥85 y) received triplet regimens as frontline therapy. More elderly pts received a single novel agent, whereas use of 2 novel agents was more common in younger pts (Table). The most common frontline regimens among elderly pts were bortezomib (V) + dexamethasone (D), followed by lenalidomide (R) + D, whereas those among younger pts included RVD, followed by VD and CyBorD (Table). No pt aged ≥85 y, and 4% of pts aged 75-84 y received high-dose chemotherapy and autologous stem cell transplant (vs 61% in the <65 y and 37% in the 65-74 y age group). The most common maintenance therapy was RD in pts ≥85 y (although the use was low) and R alone in other age groups (Table). In the ≥85 y group, 27%, 10%, and 4% of pts entered 2L, 3L, and 4L treatments respectively, vs 43%, 23%, and 13% in the <65 y group. Progression-free survival was significantly shorter in the ≥85 y age group vs the 75-84 y age group (P=0.003), 65-74 y age group (P<0.001), and <65 y age group (P<0.001; Fig.1). TTP was significantly shorter in the ≥85 y group vs the <65 y group (P=0.020); however, TTP was similar among the 65-74 y, 75-84 y, and ≥85 y cohorts (Fig. 2). Overall survival was significantly shorter in the ≥85 y group vs the 75-84 y, 65-74 y, and <65 y groups (all P<0.001; Fig. 3). The mortality rate was lowest (46%) during first-line treatment (1L) in pts aged ≥85 y (mainly attributed to MM progression) and increased in 2L and 3L (47% and 54%, respectively); a similar trend was observed in the younger age groups. The main cause of death was MM progression (29% in the ≥85 y vs 16% in the <65 y group). Other notable causes of death in the ≥85 y group included cardiac failure (5% vs 2% in <65 y group) and pneumonia (5% vs 1% in <65 y group). Conclusions: In this analysis, elderly pts received similar types of frontline and maintenance regimens as younger pts, although proportions varied with decreased use of triplet regimens with age. Considering similarities in TTP across the 65-74 y, 75-84 y, and ≥85 y cohorts, these real-world data support active treatment and aggressive supportive care of elderly symptomatic pts, including with novel agents. Additionally, further clinical studies specific to elderly patients with MM should be explored. Disclosures Lee: Amgen: Consultancy, Research Funding; GlaxoSmithKline plc: Research Funding; Sanofi: Consultancy; Daiichi Sankyo: Research Funding; Celgene: Consultancy, Research Funding; Takeda: Consultancy, Research Funding; Janssen: Consultancy, Research Funding. Ailawadhi:Janssen: Consultancy, Research Funding; Takeda: Consultancy; Pharmacyclics: Research Funding; Amgen: Consultancy, Research Funding; Celgene: Consultancy; Cellectar: Research Funding. Gasparetto:Celgene: Consultancy, Honoraria, Other: Travel, accommodations, or other expenses paid or reimbursed ; Janssen: Consultancy, Honoraria, Other: Travel, accommodations, or other expenses paid or reimbursed ; BMS: Consultancy, Honoraria, Other: Travel, accommodations, or other expenses paid or reimbursed . Jagannath:AbbVie: Consultancy; Merck & Co.: Consultancy; Bristol-Myers Squibb: Consultancy; Karyopharm Therapeutics: Consultancy; Celgene Corporation: Consultancy; Janssen Pharmaceuticals: Consultancy. Rifkin:Celgene: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees. Durie:Amgen, Celgene, Johnson & Johnson, and Takeda: Consultancy. Narang:Celgene: Speakers Bureau. Terebelo:Celgene: Honoraria; Jannsen: Speakers Bureau; Newland Medical Asociates: Employment. Toomey:Celgene: Consultancy. Hardin:Celgene: Membership on an entity's Board of Directors or advisory committees. Wagner:Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; American Cancer Society: Other: Section editor, Cancer journal. Omel:Celgene, Takeda, Janssen: Other: Patient Advisory Committees. Srinivasan:Celgene: Employment, Equity Ownership. Liu:TechData: Consultancy. Dhalla:Celgene: Employment. Agarwal:Celgene Corporation: Employment, Equity Ownership. Abonour:BMS: Consultancy; Celgene: Consultancy, Research Funding; Takeda: Consultancy, Research Funding; Janssen: Consultancy, Research Funding.

Download Full-text

Impact of Modified Thalidomide Dosing in Bortezomib/Thalidomide/Dexamethasone for Patients with Newly Diagnosed Multiple Myeloma Who Are Transplant-Eligible: A Matching-Adjusted Indirect Comparison

Blood ◽

10.1182/blood-2019-122775 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 4739-4739

Author(s):

Pieter Sonneveld ◽

Maria-Victoria Mateos ◽

Adrián Alegre ◽

Thierry Facon ◽

Cyrille Hulin ◽

...

Keyword(s):

Peripheral Neuropathy ◽

Board Of Directors ◽

Research Funding ◽

Indirect Comparison ◽

Employment Equity ◽

Advisory Committees ◽

Post Transplant ◽

Post Induction ◽

Adjusted Indirect Comparison ◽

Equity Ownership

Introduction: For patients with newly diagnosed multiple myeloma (NDMM) who are transplant-eligible, bortezomib/thalidomide/dexamethasone (VTd) is a standard of care (SoC) for induction and consolidation therapy. Clinical practice has evolved to use a modified VTd dose (VTd-mod; 100 mg thalidomide daily), which is reflected in recent treatment guidelines. As VTd-mod has become a real-world SoC, a matching-adjusted indirect comparison (MAIC) of the VTd-mod dose from recent clinical trials versus the dose included in the label (VTd-label; ramp up to 200 mg thalidomide daily) was performed to understand the effect on efficacy of modified VTd dosing for patients with NDMM who are transplant-eligible. Methods: For each outcome (overall survival [OS], progression-free survival [PFS], overall response rates [ORR] post-induction and post-transplant, and rate of peripheral neuropathy), a naïve comparison and a MAIC were performed. Data for VTd-label were obtained from the phase 3 PETHEMA/GEM study (Rosiñol L, et al. Blood. 2012;120[8]:1589-1596). Data for VTd-mod were pooled from the phase 3 CASSIOPEIA study (Moreau P, et al. Lancet. 2019;394[10192]:29-38) and the phase 2 NCT00531453 study (Ludwig H, et al. J Clin Oncol. 2013;31[2]:247-255). Patient-level data for PETHEMA/GEM and CASSIOPEIA were used to generate outcomes of interest and were validated against their respective clinical study reports; aggregate data for NCT00531453 were extracted from the primary publication. Matched baseline characteristics were age, sex, ECOG performance status, myeloma type, International Staging System (ISS) stage, baseline creatinine clearance, hemoglobin level, and platelet count. Results: Patients received VTd-mod (n = 591) or VTd-label (n = 130). After matching, baseline characteristics were similar across groups. For OS, the naïve comparison and the MAIC showed that VTd-mod was non-inferior to VTd-label (MAIC HR, 0.640 [95% CI: 0.363-1.129], P = 0.121; Figure 1A). VTd-mod significantly improved PFS versus VTd-label in the naïve comparison and MAIC (MAIC HR, 0.672 [95% CI: 0.467-0.966], P = 0.031; Figure 1B). Post-induction ORR was non-inferior for VTd-mod versus VTd-label (MAIC odds ratio, 1.781 [95% CI: 1.004-3.16], P = 0.065). Post-transplant, VTd-mod demonstrated superior ORR in both the naïve comparison and MAIC (MAIC odds ratio, 2.661 [95% CI: 1.579-4.484], P = 0.001). For rates of grade 3 or 4 peripheral neuropathy, the naïve comparison and MAIC both demonstrated that VTd-mod was non-inferior to VTd-label (MAIC rate difference, 2.4 [⁻1.7-6.49], P = 0.409). Conclusions: As naïve, indirect comparisons are prone to bias due to patient heterogeneity between studies, a MAIC can provide useful insights for clinicians and reimbursement decision-makers regarding the relative efficacy and safety of different treatments. In this MAIC, non-inferiority of VTd-mod versus VTd-label was demonstrated for OS, post-induction ORR, and peripheral neuropathy. This analysis also showed that VTd-mod significantly improved PFS and ORR post-transplant compared with VTd-label for patients with NDMM who are transplant-eligible. A limitation of this analysis is that unreported or unobserved confounding factors could not be adjusted for. Disclosures Sonneveld: Takeda: Honoraria, Research Funding; SkylineDx: Research Funding; Janssen: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; BMS: Honoraria; Amgen: Honoraria, Research Funding; Karyopharm: Honoraria, Research Funding. Mateos:Janssen, Celgene, Takeda, Amgen, Adaptive: Honoraria; AbbVie Inc, Amgen Inc, Celgene Corporation, Genentech, GlaxoSmithKline, Janssen Biotech Inc, Mundipharma EDO, PharmaMar, Roche Laboratories Inc, Takeda Oncology: Other: Advisory Committee; Janssen, Celgene, Takeda, Amgen, GSK, Abbvie, EDO, Pharmar: Membership on an entity's Board of Directors or advisory committees; Amgen Inc, Celgene Corporation, Janssen Biotech Inc, Takeda Oncology.: Speakers Bureau; Amgen Inc, Janssen Biotech Inc: Other: Data and Monitoring Committee. Alegre:Celgene, Amgen, Janssen, Takeda: Membership on an entity's Board of Directors or advisory committees. Facon:Takeda: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Amgen: Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Hulin:celgene: Consultancy, Honoraria; Janssen, AbbVie, Celgene, Amgen: Honoraria. Hashim:Ingress-Health: Employment. Vincken:Janssen: Employment, Equity Ownership. Kampfenkel:Janssen: Employment, Equity Ownership. Cote:Janssen: Employment, Equity Ownership. Moreau:Janssen: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Takeda: Consultancy, Honoraria.

Download Full-text

A Phase I Trial of Janus Kinase (JAK) Inhibition with INCB039110 in Acute Graft-Versus-Host Disease (aGVHD)

Blood ◽

10.1182/blood.v128.22.390.390 ◽

2016 ◽

Vol 128 (22) ◽

pp. 390-390 ◽

Cited By ~ 8

Author(s):

Mark A. Schroeder ◽

H. Jean Khoury ◽

Madan Jagasia ◽

Haris Ali ◽

Gary J. Schiller ◽

...

Keyword(s):

Board Of Directors ◽

Research Funding ◽

Janus Kinase ◽

Cell Transplant ◽

Employment Equity ◽

Advisory Committees ◽

Daily Dose ◽

Equity Ownership ◽

Grade 3 ◽

Steroid Refractory

Abstract Background: Corticosteroids are considered standard first-line systemic therapy for patients with aGVHD, but this approach is effective in only approximately half of all cases. For patients who progress or do not respond to corticosteroids, no specific agent has been identified as standard, and regimens are typically selected based on investigator experience and patient co-morbidities. In preclinical models, JAK inhibition has been shown to impair production of cytokines as well as the differentiation and trafficking of T cells implicated in the pathogenesis of aGVHD. Retrospective studies have suggested that JAK1/JAK2 inhibition with ruxolitinib treatment provides clinical benefit in patients with steroid-refractory GVHD (Zeiser et al, Leukemia 2015;29:2062-2068). Herein, we report preliminary safety results from a prospective randomized, parallel-cohort, open-label phase 1 trial evaluating the potent and selective JAK 1 inhibitor INCB039110 in patients with aGVHD. Methods: Male or female patients 18 years or older who underwent their first allo-hematopoietic stem cell transplant (HSCT) from any donor source and developed grades IIB-IVD aGVHD were eligible for the study. Patients were randomized 1:1 to either a 200 or 300 mg oral daily dose of INCB039110 in combination with corticosteroids, and were stratified based on prior treatment status (treatment-naive [TN] versus steroid-refractory [SR]). The primary endpoint of the study was safety and tolerability; secondary endpoints included overall response rate at Days 14, 28, 56, and 100, non-relapse mortality, and pharmacokinetic (PK) evaluations. Patients were assessed through Day 28 for dose-limiting toxicities (DLTs) and response. A Bayesian approach was used for continuous monitoring of DLTs from Days 1-28. Treatment continued until GVHD progression, unacceptable toxicity, or withdrawal from the study. Acute GVHD was graded according to MN-CIBMTR criteria; adverse events (AEs) were graded according to NCICTCAE v 4.03. Results: Between January and June 2016, 31 patients (TN, n=14; SR, n= 17) were randomized. As of July 25, 2016, data were available from 30 patients who received an oral daily dose of 200 mg (n=14) or 300 mg (n=16) INCB039110 in combination with 2 mg/kg methylprednisolone (or equivalent dose of prednisone). The median durations of treatment were 60.8 days and 56.5 days for patients receiving a daily dose of 200 mg and 300 mg INCB039110, respectively. One DLT of Grade 3 thrombocytopenia was reported. The most frequently reported AEs included thrombocytopenia/platelet count decrease (26.7%), diarrhea (23.3%), peripheral edema (20%), fatigue (16.7%), and hyperglycemia (16.7%). Grade 3 or 4 AEs occurred in 77% of patients and with similar frequency across dose groups and included cytomegalovirus infections (n=3), gastrointestinal hemorrhage (n=3), and sepsis (n=3). Five patients had AEs leading to a fatal outcome, including multi-organ failure (n=2), sepsis (n=1), disease progression (n=1), and bibasilar atelectasis, cardiopulmonary arrest, and respiratory distress (n=1); none of the fatal events was attributed to INCB039110. Efficacy and PK evaluations are ongoing and will be updated at the time of presentation. Conclusion: The oral, selective JAK1 inhibitor INCB039110 can be given safely to steroid-naive or steroid-refractory aGVHD patients. The safety profile was generally consistent in both dose groups. Biomarker evaluation, PK, and cellular phenotyping studies are ongoing. The recommended phase 2 dose will be selected and reported based on PK studies and final safety data. Disclosures Schroeder: Incyte Corporation: Honoraria, Research Funding. Khoury:Incyte Corporation: Membership on an entity's Board of Directors or advisory committees, Research Funding. Jagasia:Incyte Corporation: Research Funding; Therakos: Research Funding; Janssen: Research Funding. Ali:Incyte Corporation: Research Funding. Schiller:Incyte Corporation: Research Funding. Arbushites:Incyte Corporation: Employment, Equity Ownership. Delaite:Incyte Corporation: Employment, Equity Ownership. Yan:Incyte Corporation: Employment, Equity Ownership. Rhein:Incyte Corporation: Employment, Equity Ownership. Perales:Merck: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Seattle Genetics: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Incyte Corporation: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Chen:Incyte Corporation: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Research Funding. DiPersio:Incyte Corporation: Research Funding.

Download Full-text