scholarly journals Expression of PD-L1 in Leukemic Progenitor Cells Defines NPM1 Mutated AML As a Potential Subgroup for PD1/PD-L1 Directed Immunotherapy

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2734-2734
Author(s):  
Jochen Greiner ◽  
Vanessa Schneider ◽  
Hubert Schrezenmeier ◽  
Markus Wiesneth ◽  
Lars Bullinger ◽  
...  
Keyword(s):  
Cell Death ◽  
Programmed Cell Death ◽  
Immune Responses ◽  
Progenitor Cells ◽  
Board Of Directors ◽  
Research Funding ◽  
Significant Inhibition ◽  
Leukemic Cells ◽  
Advisory Committees ◽  
Oncology Research

Abstract Clinical and preclinical data suggest that acute myeloid leukemia (AML) with mutated nucleophosmin 1(NPM1mut) may constitute an immunogenic leukemia subtype. NPM1mut AML generally correlates with a better prognosis, however the underlying mechanisms still need to be clarified. Checkpoint inhibition targeting Programmed cell death protein 1 (PD-1)/Programmed cell death 1 ligand 1 (PD-L1) has been proven to be an effective novel immunotherapeutic approach in cancer treatment including the treatment of hematological malignancies. Expression of CD34/CD38/CD274 was evaluated in 20 NPM1mut versus 20 wild-type (NPM1wt) AML patient samples via flow cytometry analyses to assess PD-L1 (CD274) expression in leukemic cells, including leukemic progenitor and stem cells (LSC). We also investigated the influence of the anti-PD-1 antibody Nivolumab® on the antigen-specific immune responses in ELISpot assays. Additionally, we assessed the effect of Nivolumab in colony forming unit (CFU) immunoassays. Many AML cases showed relevant expression of PD-L1. Bulk cells of NPM1mut AML showed a significantly higher PD-L1 expression in comparison to NPM1wtAML patients (median of 1.5%, range 0.0-8.5%, versus 0.3%, range 0.1-1.1%). Importantly, PD-L1 expression was detected at a higher level in leukemic progenitor cells (CD34+CD38-) of NPM1mut than of NPM1wtAML (median of 3.3%, range 0.0-17.2%, versus 0.3%, range 0.0-3.0%). In general, the LSC fraction showed a higher PD-L1 expression than the non-LSC fraction. CFU immunoassays showed a significant inhibition of CFU when adding T cells stimulated against various LAA. In all patient samples, effectors activated against at least one LAA were successful to decrease the colony number significantly. Immune effects increased adding Nivolumab to the CTL for several days before starting CFU immunoassays. In summary, we detected higher PD-L1 expression in NPM1mut patients, especially in the leukemic progenitor compartment. This observation further supports the hypothesis that NPM1-directed immune responses might play an important role in tumor cell rejection, which tumor cells try to escape via expression of PD-L1. Immunogenicity of neoantigens derived from NPM1mut with higher PD-L1 expression constitute promising target structures for individualized immunotherapeutic approaches. Disclosures Schrezenmeier: Alexion Pharmaceuticals, Inc.: Honoraria, Research Funding. Bullinger:Pfizer: Speakers Bureau; Bristol-Myers Squibb: Speakers Bureau; Janssen: Speakers Bureau; Amgen: Honoraria, Speakers Bureau; Sanofi: Research Funding, Speakers Bureau; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Bayer Oncology: Research Funding. Döhner:Novartis: Consultancy, Honoraria, Research Funding; Astellas: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Celator: Consultancy, Honoraria; Jazz: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria; Celator: Consultancy, Honoraria; AROG Pharmaceuticals: Research Funding; Janssen: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; Astex Pharmaceuticals: Consultancy, Honoraria; Seattle Genetics: Consultancy, Honoraria; Pfizer: Research Funding; Seattle Genetics: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Agios: Consultancy, Honoraria; Sunesis: Consultancy, Honoraria, Research Funding; Jazz: Consultancy, Honoraria; Agios: Consultancy, Honoraria; Astellas: Consultancy, Honoraria; Pfizer: Research Funding; Celgene: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria; AROG Pharmaceuticals: Research Funding; Sunesis: Consultancy, Honoraria, Research Funding; Bristol Myers Squibb: Research Funding; Bristol Myers Squibb: Research Funding; Astex Pharmaceuticals: Consultancy, Honoraria.

scholarly journals Pre-Clinical Efficacy of Co-Treatment with KDM1A (LSD1) Inhibitor and Ruxolitinib or BET Inhibitor Against Post-MPN-sAML Blast Progenitor Cells

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1274-1274
Author(s):  
Warren Fiskus ◽  
Christopher Peter Mill ◽  
Vrajesh Karkhanis ◽  
Bernardo H Lara ◽  
Prithviraj Bose ◽  
...  
Keyword(s):  
Growth Inhibition ◽  
Progenitor Cells ◽  
Board Of Directors ◽  
Research Funding ◽  
Amine Oxidase ◽  
Genetic Alterations ◽  
Advisory Committees ◽  
Bet Inhibitor ◽  
Oncology Research ◽  
Differentiation Block

LSD1 (KDM1A) is an FAD-dependent amine-oxidase that demethylates mono and dimethyl histone H3 lysine 4 (H3K4Me1 and H3K4Me2), which regulates active enhancers and transcription in AML stem/progenitor cells (LSCs). LSD1 is part of the repressor complexes involving HDACs, CoREST or GFI1, mediating transcriptional repression and differentiation block in LSCs that persist in the minimal residual disease (MRD) following attainment of clinical complete remission, leading to relapse and poor outcome in AML. In AML LSCs, genetic alterations and epigenetic dysregulation of enhancers affect levels of myeloid transcriptional regulators, including c-Myc, PU.1, GATA 2 and CEBPα, and their target genes, which are involved in differentiation block in LSCs. Our present studies demonstrate that CRISPR/Cas9-mediated knockout of LSD1 in the AML OCI-AML5 cells significantly increased the permissive H3K4Me2/3-marked chromatin, reduced H3K27Ac occupancy at super-enhancers and enhancers (SEs/Es) (by ChIP-Seq), especially of c-Myc and CDK6, as well as repressed CoREST, c-Myc, CDK6, and c-KIT, while inducing p21, CD11b, and CD86 levels (log2 -fold change by RNA-Seq, and protein expression by Western analyses). This led to significant growth inhibition, differentiation and loss of viability of OCI-AML5 and patient-derived AML blasts (p < 0.01). Similar effects were observed following exposure of OCI-AML5 (96 hours) to tet-inducible shRNA to LSD1. Knock-down of GFI1 by shRNA (by 90%) also inhibited growth and induced differentiation, associated with upregulation of PU.1, p21 and CD11b levels. Treatment with irreversible (INCB059872, 0.25 to 1.0 µM) or reversible (SP2577, 1.0 to 2.0 µM) LSD1 inhibitor (LSD1i) inhibited binding of LSD1 to CoREST, and significantly induced growth inhibition, differentiation and loss of viability (over 96 hours) of the OCI-AML5, post-myeloproliferative neoplasm (post-MPN) sAML SET2 and HEL92.1.7 cells, as well as patient-derived AML and post-MPN sAML blasts (p < 0.01). Co-treatment with INCB059872 and ruxolitinib synergistically induced apoptosis of the post-MPN sAML SET2 and HEL92.1.7 cells and patient-derived CD34+ post-MPN sAML blasts (combination indices < 1.0). Notably, pre-treatment with the LSD1i for 48 hours significantly re-sensitized ruxolitinib-persister/resistant SET2 and HEL92.1.7 cells to ruxolitinib (p < 0.001). We previously reported that treatment with the BET inhibitor (BETi) JQ1 or OTX015 represses SE/E-driven AML-relevant oncogenes including MYC, RUNX1, CDK6, PIM1, and Bcl-xL, while inducing p21 and p27 levels in post-MPN sAML blasts (Leukemia 2017;31:678-687). This was associated with inhibition of colony growth and loss of viability of AML and post-MPN sAML blasts (p < 0.01). Here, we determined that INCB059872 treatment induced similar levels of lethality in BETi-sensitive or BETi-persister/resistant AML and post-MPN sAML cells. Since BETi treatment also depleted LSD1 protein levels, co-treatment with the BETi OTX015 and LSD1i INCB059872 or SP2577 induced synergistic lethality in AML and post-MPN sAML blasts (combination indices < 1.0). Co-treatment with INCB059872 (1.5 mg/kg) and OTX015 (50 mg/kg) both orally for 21 days, compared to each agent alone or vehicle control, significantly reduced the sAML burden and improved survival of immune-depleted mice engrafted with HEL92.1.7 or HEL92.1.7/OTX015-resistant-GFP/Luc sAML xenografts (p < 0.01). Collectively, these findings strongly support further in vivo testing and pre-clinical development of LSD1i-based combinations with ruxolitinib against post-MPN sAML and with BETi against AML or post-MPN sAML cells. Disclosures Bose: CTI BioPharma: Research Funding; Astellas: Research Funding; NS Pharma: Research Funding; Promedior: Research Funding; Constellation: Research Funding; Incyte Corporation: Consultancy, Research Funding, Speakers Bureau; Celgene Corporation: Consultancy, Research Funding; Blueprint Medicine Corporation: Consultancy, Research Funding; Kartos: Consultancy, Research Funding; Pfizer: Research Funding. Kadia:Amgen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Jazz: Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Research Funding; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees; AbbVie: Consultancy, Research Funding; Bioline RX: Research Funding; Genentech: Membership on an entity's Board of Directors or advisory committees. Bhalla:Beta Cat Pharmaceuticals: Consultancy. Khoury:Stemline Therapeutics: Research Funding; Angle: Research Funding; Kiromic: Research Funding. Verstovsek:Ital Pharma: Research Funding; Pharma Essentia: Research Funding; Astrazeneca: Research Funding; Incyte: Research Funding; CTI BioPharma Corp: Research Funding; Promedior: Research Funding; Gilead: Research Funding; Celgene: Consultancy, Research Funding; NS Pharma: Research Funding; Protaganist Therapeutics: Research Funding; Constellation: Consultancy; Pragmatist: Consultancy; Sierra Oncology: Research Funding; Genetech: Research Funding; Blueprint Medicines Corp: Research Funding; Novartis: Consultancy, Research Funding; Roche: Research Funding.

scholarly journals Rationale for and Results of a Phase I Study of the TGF-β 1/3 Inhibitor AVID200 in Subjects with Myelofibrosis: MPN-RC 118 Trial

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 6-8
Author(s):  
John Mascarenhas ◽  
Heidi E. Kosiorek ◽  
Lilian Varricchio ◽  
Rupali Bhave ◽  
Andrew T. Kuykendall ◽  
...  
Keyword(s):  
Clinical Trial ◽  
Platelet Count ◽  
Progenitor Cells ◽  
Board Of Directors ◽  
Research Funding ◽  
Normal Donor ◽  
Advisory Committees ◽  
Hematopoietic Stem ◽  
Oncology Research ◽  
Grade 3

Preclinical Rationale: Myelofibrosis (MF) is a chronic myeloproliferative neoplasm for which there are limited therapies. TGFβ plays a pivotal role in the pathobiology of MF by not only promoting bone marrow fibrosis (BMF) and collagen deposition, but also by enhancing the dormancy of normal but not MF hematopoietic stem cells (HSCs). TGFβ has also previously been reported to inhibit normal megakaryocyte (MK) production (Bruno et al Blood 1998). TGFβ1 promotes the synthesis of collagen by normal human mesenchymal stromal cells (MSCs) and activates the TGFβ receptor I/SMAD pathway as well as non-canonical TGFβ pathways. We generated MKs from MF subject mononuclear cells (MNCs) and showed that they elaborated significantly greater levels of TGFβ1 than TGFβ2/3 TGFβ1 treatment reduced the numbers of hematopoietic colonies generated by normal but not MF MNCs. Treatment of MSCs with AVID200, a potent TGFβ1/3 protein trap, significantly decreased MSC proliferation, phosphorylation of SMAD2, and collagen expression. Robust expression of pSMAD2 was observed in the absence of exogenous TGFβ in normal donor or MF-MKs, Addition of AVID200 to -MKs decreased pSMAD2 without affecting total SMAD2/3, indicating that AVID200 blocks the effects of autocrine TGFβ produced by MKs and led to increased numbers of MKs. Moreover, treatment of primary MF MNCs with AVID200 led to increased numbers of progenitor cells with wild type JAK2 and a reduction of mutated colonies. AVID200 blocked TGFβ1-induced p57Kip2 expression and SMAD2 activation by MF MNCs allowing the normal progenitor cells to preferentially cycle, proliferate, and form hematopoietic colonies. Clinical Trial Design: Based on these findings, a phase 1 trial of AVID200 is ongoing in INT-2/high risk MF subjects resistant or intolerant to ruxolitinib; baseline platelet count of ≥ 25 x 109/L, and grade 2/3 BMF. Subjects received intravenous AVID200 (Lots A and B) in dose cohorts of 180 mg/m2 (A), 550 mg/m2 (A), 180 mg/m2 (B) on Day 1 of a 21 day cycle. Cohorts of 3 subjects with a target toxicity rate of 30% were enrolled to estimate the maximum tolerated dose (MTD). A modified toxicity probability interval design was used. Response was assessed by IWG/ELN criteria after 6 cycles of AVID200. Subjects attaining at least a CI or SD with a decrease in BMF by ≥1 grade, continued AVID200. Clinical Trial Results: 10 subjects were enrolled (1 withdrew before receiving treatment) and 9 were treated with AVID200 and were evaluable for DLT assessment [Table1]. Median time after ruxolitinib discontinuation was 3.5 months (0.5-12.2). No DLTs were observed. Grade 3/4 AEs (regardless of attribution) were observed in 6 (66.7%) subjects. Grade 3/4 non-hematologic AEs observed were epistaxis (1, 11.1%), extraocular muscle paresis (1, 11.1%), fatigue (1, 11.1%) and rash (1, 11.1%). Grade 3/4 hematologic AEs were anemia (3, 33.3%) and thrombocytopenia (2, 22.2%) [Table 2]. The median number of cycles received was 5.7 (range 0 - 12). 5 subjects received 6+ cycles and were evaluable. CI occurred in 2 subjects [anemia, spleen and TSS (n=1); TSS (n=1)] 1 of which is still being treated, 2 subjects had SD, 1 subject with 21% blasts prior to study treatment had progressive MPN-BP. 4 subjects failed to reach response evaluation after 6 cycles, 2 had PD due to increasing splenomegaly, 1 subject received an allogeneic transplant and 1 is still being treated [Cycle 2]. The median platelet count at baseline was 114 (range: 42-290) and 159 after cycle 6 [Figure 1]. Maximum changes in platelets from baseline was +64% [range -73%, 169%] in all subjects. 7 subjects had an increase in platelets from baseline during treatment. 2 subjects normalized their platelet count from thrombocytopenic levels. The effect of AVID200 on BMF is currently being examined. 2 subjects remain on treatment. Conclusions: AVID200 a TGFβ1/3 protein trap is well tolerated in advanced MF subjects. Clinical responses were observed at the 550 mg dose and the expansion efficacy cohorts at doses 2 and 3 are enrolling 12 additional subjects. Furthermore, AVID200 therapy improved thrombocytopenia in MF subjects which may be due to AVID200 inhibiting the effects of TGFβ1 on normal MKpoiesis. Updated subject safety and efficacy data along with correlative data will be presented. Disclosures Mascarenhas: Celgene, Prelude, Galecto, Promedior, Geron, Constellation, and Incyte: Consultancy; Incyte, Kartos, Roche, Promedior, Merck, Merus, Arog, CTI Biopharma, Janssen, and PharmaEssentia: Other: Research funding (institution). Kuykendall:Blueprint Medicines: Research Funding; BMS: Research Funding; Incyte: Research Funding; Novartis: Research Funding. Komrokji:Jazz: Honoraria, Speakers Bureau; Abbvie: Honoraria; Agios: Speakers Bureau; BMS: Honoraria, Speakers Bureau; Geron: Honoraria; Incyte: Honoraria; Acceleron: Honoraria; Novartis: Honoraria. Gerds:Gilead Sciences: Research Funding; Imago Biosciences: Research Funding; Sierra Oncology: Research Funding; Celgene: Consultancy, Research Funding; Roche/Genentech: Research Funding; CTI Biopharma: Consultancy, Research Funding; Apexx Oncology: Consultancy; AstraZeneca/MedImmune: Consultancy; Pfizer: Research Funding; Incyte Corporation: Consultancy, Research Funding. Migliaccio:Novartis: Research Funding. O'Connor-McCourt:Forbius: Current Employment. Tremblay:Forius: Current Employment. Nadler:Forbius: Consultancy; Nadler Pharma Associates: Current Employment; Symphogen: Consultancy; Iksuda Therapeutics: Consultancy; Tessa Therapeutics: Consultancy. Mesa:Celgene: Research Funding; Genetech: Research Funding; Samus: Research Funding; Promedior: Research Funding; CTI: Research Funding; LaJolla Pharma: Consultancy; Incyte: Research Funding; Sierra Onc: Consultancy; Abbvie: Research Funding; Novartis: Consultancy. Hoffman:Forbius: Consultancy; Abbvie: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Dompe: Research Funding; Protagonist: Consultancy.

pVAX14 DNA, a Non-Specific DNA Vaccine, Improves Survival In An Acute Promyelocytic Leukemia (APL) Mouse Model Treated With All-Trans Retinoic Acid (ATRA) and Arsenic Trioxide (ATO) and Targets Leukemia Initiating Cells (LICs)

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 235-235
Author(s):  
Satyananda Patel ◽  
Laura Guerenne ◽  
Carole Le Pogam ◽  
Petra Gorombei ◽  
Patricia Krief ◽  
...  
Keyword(s):  
Mouse Model ◽  
Immune Responses ◽  
Board Of Directors ◽  
Dna Vaccine ◽  
Research Funding ◽  
Leukemic Cells ◽  
Response Monitoring ◽  
Spleen Cells ◽  
Advisory Committees ◽  
Equity Ownership

Abstract Background The combination of ATRA and ATO is very effective in treating APL, both in mouse models (Lallemand JEM 1999) and in patients (Lo Coco NEJM 2013). Having previously shown that the combination of ATRA and a specific DNA vaccine (pCDNA3PML-RARAFrC) rescues APL mice from relapse and enhances specific immune responses (Padua Nat Med 2003; Robin Blood 2006; Furugaki Blood 2010; Pokorna Mol Cell Probes 2013), we applied this approach using a novel non-specific DNA, pVAX14, as add-on therapy to ATRA +/- ATO. Methods APL transplanted mice (APL cells injected in syngeneic 6-week old mice, Brown PNAS 1997) were treated with various combinations of ATRA +/- ATO and DNA (pVAX1 (control DNA), pCDNA3PML-RARAFrC (specific DNA vaccine) or pVAX14. pVAX14 is a novel construct containing GC-rich sequences and coding for unique peptides, 3 of which we have shown to be immunogenic. Mice were monitored for survival, MRD (by RQ-PCR of PML-RARA) on days 19, 37, 57 and 67 in peripheral blood (PB), and as a measure of LICs by secondary transplantation of bone marrow (BM) or spleen cells from long term (more than 120 days) survivors (LTS) into syngeneic lethally irradiated mice. Immune responses were assessed on CD4 Memory T-cells (Tmem), IFNg production (ELISPOT) and anti-RARa (ELISA) antibody production. Functional studies including challenge experiments of LTS with leukemic cells assessed the protective effects of immunotherapy. Results 1) Survival . In a first set of experiments, ATRA+pVAX14 significantly increased survival compared to placebo, ATRA alone or ATRA+pVAX1 (control DNA) (p<0.001, p<0.03, p<0.05 respectively (Fig. 1); survival was similar with ATRA+pVAX14 and ATRA+pCDNA3PML-RARAFrC, with about 40% LTS. In a second set of experiments where ATO was introduced, ATO+pVAX14 was not superior to ATO alone, but ATRA+ATO+pVAX14 combination was significantly better than ATRA+ATO (p<0.05), providing 85% LTS (Fig. 2). MRD for PML-RARA in ATRA+ATO+pVAX14 mice was significantly lower than in ATRA mice (p<0.001) but the difference with ATRA+ATO mice was not significant up to day 67. Moreover, when we injected BM or spleen cells from both ATRA+ATO and ATRA+ATO+pVAX14 LTS none of the recipients develop APL (follow-up period of 140 days), indicating the clearance of LICs. 2) Immune response monitoring showed that effector Tmem were significantly higher in ATRA+ATO+pVAX14 > ATRA+ATO > ATRA (p<0.05 and p<0.001 respectively). LTS of ATRA+ATO+pVAX14 had increased IFNg producing lymph node cells when stimulated with APL cells or PML-RAR peptide compared to ATRA+ATO (p<0.02). Serum anti-RARa antibodies were significantly increased in all combinations compared to controls with ATRA+ATO+pVAX14 > ATRA+pVAX14 ≈ ATRA+ATO+pVAX1 (control DNA) ≈ ATRA+ATO > ATRA+pVAX1 (Control DNA) > ATRA (p<0.05, p<0.03, p<0.0006 respectively). When LTS mice of ATRA+ATO+pVAX14 and ATRA+ATO groups were challenged with APL spleen cells, survival was significantly higher in both groups (median survival 105 vs 80 days, p=NS) than in controls (syngeneic mice injected with APL cells) (p<0.001 and p<0.01 respectively) Conclusion The combination of the non-specific vaccine (pVAX14) with ATRA+ATO further improved survival over ATRA+ATO alone and elicited specific humoral and T-cell mediated responses targeting LICs. This vaccine is also effective in a mouse model of high risk MDS (Submitted to ASH 2103). SP, LG and CLP contributed equally to this work. Disclosures: Patel: CEFIPRA: Research Funding. Fenaux:Celgene: Honoraria, Research Funding. Chomienne:Vivavacs: Equity Ownership, I have patents pending through INSERM/Paris-Diderot related to technology employed in this present study. , I have patents pending through INSERM/Paris-Diderot related to technology employed in this present study. Patents & Royalties, Membership on an entity’s Board of Directors or advisory committees. Padua:Vivavacs: Equity Ownership, I have patents pending through INSERM/Paris-Diderot related to technology employed in this present study. I, I have patents pending through INSERM/Paris-Diderot related to technology employed in this present study. I Patents & Royalties, Membership on an entity’s Board of Directors or advisory committees.

scholarly journals Inhibition of Mcl-1 Enhances the Efficacy of Tyrosine Kinase Inhibition in FLT3 Mutated AML and Synergizes with Venetoclax Targeting AML Stem Cells

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1267-1267 ◽  
Author(s):  
Bing Z Carter ◽  
Wenjing Tao ◽  
Po Yee Mak ◽  
Qi Zhang ◽  
Xiangmeng Wang ◽  
...  
Keyword(s):  
Cell Death ◽  
Tyrosine Kinase ◽  
Progenitor Cells ◽  
Board Of Directors ◽  
Median Survival ◽  
Research Funding ◽  
Cancer Center ◽  
Advisory Committees ◽  
Equity Ownership

Bcl-2 and Mcl-1 play critical roles in AML stem/progenitor cell survival. Venetoclax (VEN), a highly selective Bcl-2 inhibitor, showed limited clinical efficacy in AML as a single agent. FLT3 is the most frequently mutated gene in AML, resulting in constitutive activation of FLT3 tyrosine kinase and its downstream signaling pathways, which can be targeted by FLT3 tyrosine kinase inhibitors (TKIs). However, patients can adapt to TKI treatment by reactivating the MEK signaling pathway (Bruner JK et al., Cancer Res 2017), which is known to stabilize Mcl-1 levels. Furthermore, deregulated Mcl-1 expression was identified as a novel mechanism of primary TKI resistance in a subset of FLT3-ITD mutated AML patients (Breitenbuecher F et al., Blood 2009). Importantly, Mcl-1 can be induced by VEN treatment and could be a major resistance factor to VEN (Pan R et al., Cancer Discover 2014; Carter BZ et al., ASH 2018). Hence, Mcl-1 inhibition may enhance the efficacy of TKIs in FLT3 mutated AML and synergize with VEN, targeting AML cells and stem/progenitor cells. We treated FLT3-ITD positive AML cells with a selective inhibitor of Mcl-1 (AMG 176) and FLT3 TKIs and found that inhibition of Mcl-1 induced cell death and significantly enhanced the activity of sorafenib or gilteritinib in cell lines including cells acquired resistance to VEN (CI<1). It also enhanced the activity of sorafenib against blasts and stem/progenitor cells from primary AML samples harboring FLT3-ITD mutations. We previously showed that overexpression/knockdown of Mcl-1 greatly protected/sensitized AML cells from VEN induced cell death (Carter BZ, ASH 2018) supporting Mcl-1 as a key VEN resistance factor. We treated primary AML cells (n=5) with VEN (10 nM) or AMG 176 (250 nM) alone, or in combination and found that VEN+AMG 176 synergistically induced cell death in AML blasts and AML stem/progenitor cells even in samples clinically resistant to or relapsed after VEN containing regimen (CI<1). This synergism was also observed under mesenchymal stromal cells co-culturing conditions, while the combination was less toxic to normal bone marrow (NBM) cells (n=3) at even higher concentrations (VEN 20 nM, AMG 176 500 nM): apoptosis rate was at 82.4% or 80.8% under MSC co-cultures with AML blasts vs 34.2% or 36.4% under co-culture with NBM CD34+ cells. To investigate the antileukemia activity in vivo, we tested combined inhibition of Mcl-1 and Bcl-2 using two PDX models in NSG mice. The first model was developed from a resistant/relapsed patient with FLT3-ITD mutation and complex karyotype. The combination showed the most significant antileukemic activity and extension of survival, followed by AMG 176 and VEN treatment alone (median survival for the combination, 146 d, p=0.004; AMG 176, 137 d, p=0.032; VEN, 102 d, p>0.05 vs. control, 85.5 d; respectively). The second PDX model was developed from a patient who first responded and then became resistant to the combination of VEN and decitabine and harbors FLT3-ITD, NRAS, and GATA2 mutations and complex karyotype. VEN or AMG 176 monotherapies marginally prolonged survival (median survival 127 or 129 vs. control 124 d). The combination was highly effective in this model and greatly decreased circulating blasts (Fig. 1) and leukemia tissue infiltration, measured by flow cytometry and spleen size. CyTOF analysis demonstrated that only the combination strongly reduced blasts as well as the AML stem/progenitor cell populations. Median survival for the combination group currently has not been reached (>325 d) (Fig. 2). Collectively, these data demonstrate that inhibition of Mcl-1 enhances the efficacy of TKIs in FLT3 mutated AML. Furthermore, it synergizes with VEN, targeting not only AML blasts but also AML stem/progenitor cells, both in vitro and in vivo in PDX models with the potential of significantly improving treatment outcome, which warrants clinical evaluation. Disclosures Carter: Amgen: Research Funding; AstraZeneca: Research Funding; Ascentage: Research Funding. Zhang:The University of Texas M.D.Anderson Cancer Center: Employment. Kuruvilla:The University of Texas M.D.Anderson Cancer Center: Employment. Konopleva:Kisoji: Consultancy, Honoraria; Eli Lilly: Research Funding; Forty-Seven: Consultancy, Honoraria; Calithera: Research Funding; Stemline Therapeutics: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria, Research Funding; Cellectis: Research Funding; Reata Pharmaceuticals: Equity Ownership, Patents & Royalties; Amgen: Consultancy, Honoraria; F. Hoffman La-Roche: Consultancy, Honoraria, Research Funding; Genentech: Honoraria, Research Funding; Ascentage: Research Funding; Ablynx: Research Funding; Agios: Research Funding; Astra Zeneca: Research Funding. Caenepeel:Amgen Inc.: Employment. Canon:Amgen Inc.: Employment. Hughes:Amgen Inc.: Employment. Morrow:Amgen Inc.: Employment. Andreeff:Daiichi Sankyo, Inc.: Consultancy, Patents & Royalties: Patents licensed, royalty bearing, Research Funding; Celgene: Consultancy; Jazz Pharmaceuticals: Consultancy; Amgen: Consultancy; AstaZeneca: Consultancy; 6 Dimensions Capital: Consultancy; Reata: Equity Ownership; Aptose: Equity Ownership; Eutropics: Equity Ownership; Senti Bio: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Oncoceutics: Equity Ownership; Oncolyze: Equity Ownership; Breast Cancer Research Foundation: Research Funding; CPRIT: Research Funding; NIH/NCI: Research Funding; 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; NCI-CTEP: Membership on an entity's Board of Directors or advisory committees; 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; 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; BiolineRx: Membership on an entity's Board of Directors or advisory committees.

scholarly journals VTE Rates and Safety Analysis of Newly Diagnosed Multiple Myeloma Patients Receiving Carfilzomib-Lenalidomide-Dexamethasone (KRD) with or without Rivaroxaban Prophylaxis

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1835-1835 ◽  
Author(s):  
Katrina M Piedra ◽  
Hani Hassoun ◽  
Larry W. Buie ◽  
Sean M. Devlin ◽  
Jessica Flynn ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Venous Thromboembolism ◽  
Board Of Directors ◽  
Research Funding ◽  
High Dose ◽  
Newly Diagnosed ◽  
Advisory Committees ◽  
Cancer Trial ◽  
Oncology Research ◽  
Increased Risk

Introduction Immunomodulatory agents (IMiD's) are associated with an increased risk of venous thromboembolism (VTE), particularly when combined with high dose steroids. Studies evaluating the use of lenalidomide-bortezomib-dexamethasone (RVD) and carfilzomib-lenalidomide-dexamethasone (KRD) in the frontline setting for multiple myeloma (MM) have reported a 6% and 24% incidence of thrombosis, respectively, despite primary thrombotic prophylaxis with aspirin (ASA) (Richardson, et al. Blood. 2010; Korde, et al. JAMA Oncol 2015). Recent data, including the Hokusai VTE Cancer Trial, have suggested that safety and efficacy of direct oral anticoagulants (DOACs) are preserved in the setting of treatment of solid malignancy-associated thrombosis (Raskob, et al. N Engl J Med. 2018; Mantha, et al. J Thromb Thrombolysis. 2017). Despite this data, there is limited experience and use of DOACs in prevention of thromboses in the setting of hematologic malignancies, specifically MM. After careful review of literature, since early 2018, we changed our clinical practice and routinely placed newly diagnosed MM (NDMM) patients receiving KRD at Memorial Sloan Kettering Cancer Center (MSKCC) on concomitant rivaroxaban 10 mg once daily, regardless of VTE risk stratification. In the following abstract, we present VTE rates and safety data for newly diagnosed MM patients receiving RVD with ASA vs. KRD with ASA vs. KRD with rivaroxaban prophylaxis. Methods This was an IRB-approved, single-center, retrospective chart review study. All untreated patients with newly diagnosed MM, receiving at least one cycle of RVD or KRD between January 2015 and October 2018 were included. The period of observation included the time between the first day of therapy until 90 days after completion of induction therapy. Patients were identified by querying the pharmacy database for carfilzomib or bortezomib administration and outpatient medication review of thromboprophylaxis with rivaroxaban or ASA. VTE diagnoses were confirmed by ICD-10 codes and appropriate imaging studies (computed tomography and ultrasound). Descriptive statistics were performed. Results During the observation period, 241 patients were identified to have received RVD or KRD in the frontline (99 RVD with ASA; 97 KRD with ASA; 45 KRD with rivaroxaban). Baseline characteristics were well distributed among the three arms, with a median age of 60 (30-94) in the RVD ASA arm, 62 (33-77) in the KRD ASA arm, and 60 (24-79) in the KRD rivaroxaban arm. Patients had International Staging System (ISS) stage 3 disease in 13% (N=13), 9.3% (N=9), and 11% (N=5) of the RVD ASA, KRD ASA, and KRD rivaroxaban arms, respectively. Median weekly doses of dexamethasone were higher in both KRD arms, 40 mg (20-40) vs. 20 mg (10-40) in the RVD ASA arm. The average initial doses of lenalidomide were 22 mg in the RVD ASA arm compared to 25 mg in both the KRD ASA and KRD rivaroxaban arms. After querying the pharmacy database, no patients were identified to have a history or concomitant use of erythropoietin stimulating agent (ESA) use. Treatment-related VTE's occurred in 4 patients (4.0%) in the RVD ASA arm, 16 patients (16.5%) in the KRD ASA arm, and in 1 patient (2.2%) in the KRD rivaroxaban arm. Average time to VTE was 6.15 months (Range 5.42, 9.73) after treatment initiation in the RVD ASA group, while it was 2.61 months (Range 0.43, 5.06) in the KRD ASA group and 1.35 months in the KRD rivaroxaban group. Minor, grade 1 bleeding events per the Common Terminology Criteria for Adverse Events (CTCAE) were identified in 1 (1.1%) patient in the RVD ASA arm, 5 (5.2%) patients in the KRD ASA arm, and 1 (2.2%) patient in the KRD rivaroxaban arm. Conclusion More efficacious MM combination therapies have been found to increase the risk of VTE when using ASA prophylaxis, indicating better thromboprophylaxis is needed. We found patients receiving ASA prophylaxis with KRD were more likely to experience a VTE and these events occurred earlier compared to patients receiving ASA prophylaxis with RVD. Importantly, the rate of VTE was reduced to the same level as ASA prophylaxis with RVD when low-dose rivaroxaban 10 mg daily was used with KRD, and without necessarily increasing bleeding risk. Our retrospective data support the development of prospective clinical trials further investigating DOAC use in thromboprophylaxis for NDMM patients receiving carfilzomib-based treatments. Figure Disclosures Hassoun: Novartis: Consultancy; Janssen: Research Funding; Celgene: Research Funding. Lesokhin:BMS: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria; Janssen: Research Funding; GenMab: Consultancy, Honoraria; Serametrix Inc.: Patents & Royalties; Genentech: Research Funding; Juno: Consultancy, Honoraria. Mailankody:Juno: Research Funding; Celgene: Research Funding; Janssen: Research Funding; Takeda Oncology: Research Funding; CME activity by Physician Education Resource: Honoraria. Smith:Celgene: Consultancy, Patents & Royalties, Research Funding; Fate Therapeutics and Precision Biosciences: Consultancy. Landgren:Theradex: Other: IDMC; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Other: IDMC; Sanofi: Membership on an entity's Board of Directors or advisory committees; Adaptive: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. OffLabel Disclosure: Off-label use of rivaroxaban for outpatient prophylaxis of venous thromboembolism (VTE) will be explicitly disclosed to the audience.

scholarly journals Risk Factors Associated with the Development of Infusion-Related Reactions in Patients with Chronic Lymphocytic Leukaemia Treated with Anti-CD20 Monoclonal Antibodies: Analysis of the CLL11 Study Dataset

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3339-3339 ◽  
Author(s):  
Ciara Louise Freeman ◽  
Mark Dixon ◽  
Richard Houghton ◽  
Kathryn Humphrey ◽  
Gunter Fingerle-Rowson ◽  
...  
Keyword(s):  
Risk Factors ◽  
Monoclonal Antibodies ◽  
Board Of Directors ◽  
Research Funding ◽  
Lymphocytic Leukaemia ◽  
Signs And Symptoms ◽  
Leukemic Cells ◽  
Tumour Burden ◽  
Advisory Committees ◽  
Anti Cd20

Abstract Background: The administration of anti-CD20 monoclonal antibodies (mAb) in patients with B-cell lympho-proliferative disorders is frequently accompanied by a constellation of signs and symptoms that have been labelled as infusion-related reactions (IRR). The pathophysiology of IRR remains poorly understood as do predictors of risk, which may relate to the mechanism of action of the anti-CD20, disease-related factors such as tumour burden or host factors such as polymorphisms of Fc gamma receptor 3A (FcγRIIIA). In the CLL11 trial (NCT01010061), patients with previously untreated chronic lymphocytic leukaemia and comorbidities were randomised to receive either rituximab (type I anti-CD20 mAb) or obinutuzumab (type II and glycoengineered anti-CD20 mAb) in combination with chlorambucil for six cycles. Obinutuzumab led to faster depletion of B cells and achieved an improvement in outcome parameters such as response and progression-free survival compared with the rituximab arm, but was also associated with a higher rate and increased severity of IRR. To better understand the profile of risk for IRR in patients with CLL, we performed an exploratory analysis on data obtained from patients treated with either one of the two antibodies given in combination with chlorambucil. Methods: Patients from the prospective, randomized Phase III CLL11 study who received a first infusion of obinutuzumab (N=331) or rituximab (N=326) were included. Baseline pre-treatment risk factors thought to play a possible role in the development of IRR were identified a priori and included patient demographics, concurrent conditions and premedications, parameters of disease burden, prognostic factors, laboratory variables and FcγR genotype. Baseline values for mean fluorescence intensity (MFI) of CD20, gated on the circulating CLL clone, and MFI of CD16, gated on the natural killer (NK) cell population (CD56+16+) in peripheral blood were also available for N=510 patients. The primary outcome, development of an IRR with the first infusion, was defined as the occurrence of related signs and symptoms during or within 24 hours of administration of antibody. Due to the short-term nature of the initial IRR a multivariate logistical regression analysis was performed rather than a time to event analysis. Internal validation of this model, derived from a single dataset, was conducted using the established resampling technique of bootstrapping. This assessed the proportion of times each variable retained significance at α=0.10 when the model was fitted to bootstrapped samples of the dataset. Results: Patients that appeared to be at greater risk of developing any grade of IRR with the first infusion of rituximab or obinutuzumab were those treated with obinutuzumab, those with higher surface expression CD20 on CLL cells (MFI CD20) and greater FcγRIIIA (MFI CD16) on NK cells in peripheral blood, those with higher affinity FcγRIIIA genotype (VV), more pronounced neutropenia and splenomegaly at baseline (Table 1). Higher baseline absolute lymphocyte count and the presence of respiratory comorbidity also appeared to increase risk. All variables significant for inclusion in the model are shown in Table 1. Looking at those patients treated with obinutuzumab only, the most important determinant of risk was MFI CD20 (OR 3.6 95% CI 1.6-7.9). The impact of glucocorticoid premedication in reducing risk in obinutuzumab treated patients was not sufficient to reach significance, however, patients were not randomised to this intervention. Conclusion: This work identifies novel disease- and patient-specific biological variables that appear to play a role in the development of IRR in patients with CLL treated with anti-CD20 mAb, although the treatment received (obinutuzumab >rituximab) confers greatest risk. In addition to parameters of tumour burden, target antigen expression and gene polymorphisms of FcγR also appear to contribute to the risk of developing an IRR. Our results support the hypothesis that higher rates of IRR seen with the administration of obinutuzumab may result from stronger activation upon binding to CD20 on leukemic cells and subsequent enhanced cross-linking between CD20 expressing leukemic cells and FcγRIIIA bearing effector cells. Further studies involving obinutuzumab in this patient population will be needed to externally validate the results of this exploratory analysis. Disclosures Freeman: Roche Pharmaceuticals: clinical research fellowship supported by Roche Pharmaceuticals (secondment from Bart's) Other. Dixon:Roche Pharmaceuticals: Employment. Houghton:Roche Pharmaceuticals: Employment. Humphrey:Roche: Employment. Fingerle-Rowson:Roche Pharmaceuticals: Employment. Kreuzer:Roche Pharmaceuticals: Research Funding. Engelke:Roche: Travel grants Other. Hallek:Roche Pharmaceuticals: Consultancy, Research Funding, Speakers Bureau. Goede:Bristol Myers Squibb: Honoraria; Mundipharma: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Travel grants Other.

IGHV Unmutated Chronic Lymphocytic Leukemia (CLL) B Cells Are More Susceptible to Spontaneous Apoptosis Than Mutated CLL B Cells and Are Subject to the Anti-Apoptotic Effect of Environmental Signals

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2431-2431
Author(s):  
Marta Coscia ◽  
Francesca Pantaleoni ◽  
Chiara Riganti ◽  
Candida Vitale ◽  
Micol Rigoni ◽  
...  
Keyword(s):  
T Cells ◽  
B Cells ◽  
Board Of Directors ◽  
Stromal Cells ◽  
Peripheral Blood ◽  
Research Funding ◽  
Lymphocytic Leukemia ◽  
Leukemic Cells ◽  
Spontaneous Apoptosis ◽  
Advisory Committees

Abstract Abstract 2431 Chronic lymphocytic leukemia (CLL) is a clinically heterogeneous disease. A very reliable prognosticator is the mutational status of the tumor immunoglobulin heavy chain variable region (IGHV): patients with unmutated (UM) IGHV have a worse prognosis than patients with mutated (M) IGHV. Soluble factors (i.e. IL-4 and CD40L) and cellular components of the local microenvironment [i.e. bone marrow stromal cells (BMSC) and nurse-like cells (NLCs)] are important survival factors for CLL B cells. It is currently unknown to what extent UM and M CLL cells depend on the local microenvironment for their survival. We have evaluated the spontaneous apoptotic rate of tumor cells isolated by immunomagnetic selection from the peripheral blood (PB) of M and UM CLL patients. Both M and UM CLL B cells underwent spontaneous apoptosis throughout the culture period. However, the UM CLL B cells showed a significantly higher degree of apoptosis in 7-day cultures as compared to M CLL B cells. In both M and UM CLL B cells, high basal levels of Bcl-2 expression and NF-kB activity were detected. On day 7, the percentage of Bcl-2+ leukemic cells was significantly lower in UM than in M CLL B cells. EMSA test showed that NF-kB was totally inactivated in UM CLL B cells and only partially reduced in M CLL B cells. Quantitative analysis of RelA and RelB subunits showed that NF-kB inactivation in UM CLL B cells consisted in a strong reduction of both RelA and RelB nuclear expression. CD40L, IL-4 and stromal cells significantly improved UM CLL B cells viability and significantly recovered Bcl-2 expression. The protective effect exerted by these stimuli was totally independent from the recovery of NF-kB expression. Indeed, after 7 days of culture, the UM CLL B cells had completely lost the nuclear form of NF-kB, and none of the stimuli was capable of restoring it. We observed that UM CLL cells were less susceptible to spontaneous apoptosis when cultured as unfractionated peripheral blood mononuclear cells (M or UM PBMC) as compared to purified leukemic cells (M and UM CLL B cells). The reduced apoptosis detected in UM PBMC was accompanied by a retained expression of Bcl-2 and by a restored activity of NF-kB and suggested the presence of a pro-survival element in the peripheral blood of these patients. To investigate the role of NLC in rescuing UM CLL B cells from apoptosis we first evaluated whether M and UM PBMC generated NLC with the same efficiency. Unexpectedly, the former generated significantly higher numbers of NLC than UM PBMC. Despite the lack of generation of NLC, CLL B cells viability was very similar in the non-adherent fraction of M and UM PBMC on day 7 and 14 of culture. This observation ruled out a role for NLC in supporting UM CLL B cells survival. Conversely, a pro-survival effect on UM CLL B cells was exerted by autologous T cells. Indeed, a significant reduction in the apoptotic rate of leukemic cells was observed when purified UM CLL B cells were cultured in the presence of autologous peripheral blood T cells (UM CLL B cell/T cell co-cultures). NF-kB activity was completely lost in UM CLL B cells cultured for 7 days in medium alone whereas it was restored in UM CLL B cells / T cells co-cultures. The prosurvival effect of circulating T cells was exerted both in cell-to-cell contact and in trans-well condition and was associated to increased secretions of tumor necrosis factor-alpha (TNF-α), platelet-derived growth factor (PDGF)-BB and interleukin-8 (IL-8) as detected by analyses of supernatants through a Multiplex system. These data indicate that despite their more aggressive features, UM CLL B cells are more susceptible to spontaneous apoptosis and depend from environmental prosurvival signals. This vulnerability of UM CLL B cells can be exploited as a selective target of therapeutic interventions. Disclosures: Boccadoro: Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen-Cilag: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Massaia: Novartis: Honoraria, Research Funding.

Preliminary Results of a Phase 2 Study of PD 0332991 in Combination with Bortezomib and Dexamethasone in Patients with Relapsed and Refractory Multiple Myeloma

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2940-2940
Author(s):  
Ruben Niesvizky ◽  
Luciano J Costa ◽  
Nisreen A. Haideri ◽  
Georg Hess ◽  
Seema Singhal ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Board Of Directors ◽  
Research Funding ◽  
Phase 1 ◽  
Phase 2 ◽  
Advisory Committees ◽  
Oncology Research ◽  
Equity Ownership ◽  
D 20 ◽  
Ecog Ps

Abstract Abstract 2940 Background: PD 0332991 is an orally bioavailable selective inhibitor of cyclin-dependent kinase (CDK) 4/6. Inhibition of CDK4/6 phosphorylation of retinoblastoma (Rb) induces prolonged early G1 cell cycle arrest (pG1) and synchronous progression to S phase (pG1-S) upon withdrawal, which sensitizes human multiple myeloma (MM) cells to killing by bortezomib (B) or dexamethasone (D) in vitro and in animal models. Based on these observations, a phase 1/2 study in combination with B plus D in patients (pts) with relapsed and/or refractory MM was initiated. The phase 1 part of the study (completed) determined the recommended phase 2 dose and schedule to be PD 0332991 100 mg QD 12 days on followed by 9 days off treatment in a 21-day cycle with intravenous B 1.0 mg/m2 plus oral D 20 mg administered on Days 8 and 11 in pG1 and 15 and 18 in pG1-S (Niesvizky et al. ASH 2010). We present preliminary data from the phase 2 part of the study. Methods: Pts with Rb protein-positive, measurable (as defined by International Myeloma Working Group [IMWG]) progressive, relapsed or refractory MM after ≥1 prior treatment were eligible. Prior B was allowed only if there was a response and disease progression occurred off therapy. Pts received oral PD 0332991 once daily on Days 1–12 in a 21-day cycle in combination with intravenous B 1.0 mg/m2 plus oral D 20 mg administered on Days 8, 11, 15, and 18. The primary endpoint is overall response rate (ORR); secondary endpoints include time to progression (TTP), progression-free survival (PFS), overall survival, duration of response, and safety. PD 0332991-mediated inhibition of CDK4/6-specific phosphorylation of Rb (pSRb) and Ki67 in bone marrow MM cells were also assessed. The phase 2 part of the study is a Simon Two-Stage Minimax design; 25 response evaluable patients were to be enrolled into the first stage. Results: 39 pts have been tested for Rb and 36 pts (92%) were positive. Of the 36 pts, 30 pts have been enrolled to date including 2 pts who did not receive the study treatment, and 23 pts are considered response evaluable as of the data cut-off. 56% of pts had an Eastern Cooperative Oncology Group performance status (ECOG PS) of 1 and 8% had ECOG PS of 2. At baseline, median β2 microglobulin was 3.1 (range 1.6–26.2), median hemoglobin was 11.2 (7.2–13.6), median calcium was 9.4 (8.7–11.9). The median number of prior therapies was 2 (range 1–8); 55% had received prior B. Sixteen pts have discontinued (9 due to progressive disease, 3 due to AE, 2 consent withdrawal, and 2 not treated). The most common treatment-related AEs were thrombocytopenia (44%), nausea (20%), anemia, constipation, fatigue, and neutropenia (all 16%); 32% of pts reported grade ≥3 thrombocytopenia. IHC data showed on-treatment reduction in pSRb and Ki67 in MM cells from bone marrow of 3/3 patients with available samples. To date, 1 pt achieved a complete response (CR), 1 achieved a very good partial response (VGPR), 1 partial response (PR), 1 minor response (MR), and 5 stable disease (SD); 6 pts are too early for assessment. Conclusions: To date, the combination of PD 0332991 and B plus D has shown response in 4 pts with relapsed/refractory MM. The most commonly reported AEs were cytopenias, consistent with the known safety profiles of PD 0332991 and B. PD 0332991 inhibited phosphorylation of Rb and cell cycle progression in MM cells. The accrual to stage 1 is ongoing. Updated efficacy and safety data will be presented. Disclosures: Niesvizky: Millennium Pharmaceuticals: Consultancy; Millennium Pharmaceuticals: Research Funding; Millennium Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Hess:Pfizer Oncology: Consultancy; Pfizer Oncology: Research Funding; Pfizer Oncology: Membership on an entity's Board of Directors or advisory committees. Spicka:Janssen-Cilag: Consultancy; Celgene: Consultancy; Celgene: Research Funding; Janssen-Cilag: Honoraria; Celgene: Honoraria; Janssen-Cilag: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees. Jakubczak:Pfizer Oncology: Employment; Pfizer Oncology: Equity Ownership. Kim:Pfizer Oncology: Equity Ownership; Pfizer Oncology: Employment. Randolph:Pfizer Oncology: Employment; Pfizer Oncology: Equity Ownership. Chen-Kiang:Pfizer Oncology: Research Funding.

Functional Defects In Neutrophils Derived From Ezh2 Null Mice

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1556-1556
Author(s):  
Albert Perez-Ladaga ◽  
Bennett Caughey ◽  
Huafeng Xie ◽  
Stuart H. Orkin ◽  
David B. Sykes ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Death ◽  
Progenitor Cells ◽  
Board Of Directors ◽  
Cell Lines ◽  
Resting Cells ◽  
Surface Markers ◽  
Advisory Committees ◽  
Spontaneous Cell Death

Abstract Introduction We investigate the role of Ezh2 in neutrophil function using murine progenitor cells differentiated into neutrophils lacking the Ezh2 gene. Ezh2 is the catalytic component of the polycomb repressive complex 2, which methylates lysine 27 of histone H3. It is frequently disrupted in myelodysplastic syndromes (MDS) leading to loss of function (Ernst et al., 2010). Mutations in EZH2 are found in 6% of MDS patients and while not strongly linked to cytopenias or blast proportion, they are independently associated with worse overall survival compared to patients with wildtype EZH2 (Bejar R. et al., 2011 and 2012). We hypothesize that Ezh2 mutations may cause qualitative defects in myeloid cells that impact their function and could contribute to the adverse prognosis observed in EZH2 mutant MDS. Methods Bone marrow from Ezh2 null (Ezh2-/-) and littermate control mice (WT) were transduced with HOXB8 fused to the estrogen receptor ligand-binding domain to produce immortalized myeloid progenitor cells. Removal of estrogen from the media allows these cells differentiate into mature neutrophils (Wang G.G., 2006). Differentiated cells were characterized for surface markers by flow cytometry and for gene expression by PCR of mRNA. Spontaneous cell death was measured by annexin/PI staining. Cell cycle patterns were determined by measuring the red emission of PI. Chemotactic function was assessed by counting cells that migrated across a transwell in presence/absence of the attractant zymosan. For phagocytosis experiments, cells were incubated with Fluoresbrite YG carboxylate beads at 37°C or 4°C. Reactive oxygen species (ROS) generation was measured by the oxidation of dihydrorhodamine 123 into fluorescent rhodamine 123. Results Estrogen withdrawal caused differentiation of both WT and Ezh2-/- lines into cells with mature neutrophil morphology after six days (Figure 1a). Both differentiated lines expressed the neutrophil surface markers CD11b and CD62L and the neutrophil-specific genes lactoferrin and Itgb2l. Ezh2 -/- cells had an increased rate of spontaneous cell death compared to WT in undifferentiated (32.81% vs. 20.33%) and mature cells (32.82% vs. 14.23%). Nevertheless, both progenitor cell lines showed similar cell cycle patterns, demonstrating that Ezh2 absence had no other effect on cell cycle progression. Ezh2 -/- neutrophils failed to migrate towards zymosan (Figure 1b). Expression of Tlr2, which binds zymosan, and other Toll-like receptors (Tlr4/5/9) were similar between the differentiated cell lines. Cells incubated with FITC-zymosan at 37°C showed no fluorescence differences between cell lines, indicating similar adherence. Experiments with neutrophils from an MDS patient with homozygous EZH2 mutations demonstrated a similar migration defect. Additional studies in MDS patient samples are ongoing and will be presented. Phagocytosis was reduced in Ezh2-/-cells. Unstimulated, the number of cells ingesting and adhering YG-beads was significantly greater with WT cells than with Ezh2-/-cells. When activated with fMLP, both lines showed increased adherence of YG-beads but the number of phagocytosing Ezh2-/- cells was reduced. The average number of beads ingested by each cell was lower for Ezh2-/- cells compared to WT (5.95 vs 2.94, p < 0.001) in resting cells, and 9.47 vs. 3.73 in fMLP-activated cells, p < 0.01. The fraction of Ezh2-/- neutrophils generating ROS when stimulated with PMA is 2.4-fold higher than for WT cells. ROS production was greatly reduced in the presence of diphenyleneiodonium (DPI), confirming the role of NADPH oxidase in the generation of ROS. Conclusion Our results indicate impaired function of neutrophils derived from Ezh2-/- mice, demonstrating increased spontaneous cell death, impaired migration, decreased phagocytosis, and overproduction of ROS. Qualitative defects observed in neutrophils deficient for EZH2 may help explain the adverse prognosis associated with these mutations in MDS patients. Disclosures: Bejar: Genoptix: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity’s Board of Directors or advisory committees.

FLT3 Splice Variant (FLT3Va) As a Potential Immunotherapeutic Target in Patients with Acute Myeloid Leukemia (AML)

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1681-1681
Author(s):  
Sophia Adamia ◽  
Jeffrey Nemeth ◽  
Shruti Bhatt ◽  
Sarah R Walker ◽  
Natalie I Voeks ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Surface ◽  
Board Of Directors ◽  
Research Funding ◽  
Leukemic Cells ◽  
Advisory Committees ◽  
Gm Csf ◽  
Nsg Mice ◽  
Blocking Antibodies ◽  
Pdx Models

Abstract Alternative pre-mRNA splicing (AS) is a normal epigenetic phenomenon, a key regulator of gene expression, yields multiple transcripts and thus a variety of proteins from a single gene. Mutations in the spliceosome components resulting in aberrant splicing isoforms are common in AML, and other myeloid neoplasms, and may generate leukemia-specific neoantigens targetable with an antibody-drug conjugates (ADCs) or blocking antibodies. Our previous studies revealed that the FLT3 cell surface receptor is one of the most commonly misspliced genes in AML (54-63% of ~400 AML patients). We conducted cloning and sequencing analyses in AML cells and identified multiple aberrant splice-variants of FLT3 that resulted from either skipping of one or more exons or activation of cryptic splicing sites. Transfection of cDNA with three of these variants in TF-1 (AML cell line) cells resulted in expression of Flt3 variant proteins on the cell surface. We successfully generated rabbit polyclonal antiserum against a unique peptide sequence present in the most commonly expressed abnormal splice variant, which we termed Flt3Va. Immunoblots performed with the polyclonal antibody identified a ~160 kDa protein expressed by TF-1 cells transfected with FLT3Va, and the antibody did not react with untransfected TF-1 cell lysate. Using standard techniques, we generated rabbit hybridomas and evaluated the clones by flow cytometry and western blotting experiments. Based on these data, we selected one antibody clone (15-7) for further experiments. The 15-7 anti-Flt3Va rabbit monoclonal antibody identified Flt3Va protein expressed on the cell surface and within the cytoplasm of transfected TF-1 cells by flow cytometry and western blotting. However, no Flt3Va protein was detected in untransfected TF-1 cells or normal CD34+ bone marrow cells. The 15-7 antibody bound to 26 of 52 primary AML samples and 5 of 10 primagraft samples (PDX models) of human AML. Immunoblotting analyses of PDX models and patient samples confirmed binding to a protein of the expected size (130-160 kDa). Additionally, multi-parameter flow cytometry in 10 PDX models and 52 primary demonstrated that putative AML stem cells (as defined by the CD45dim, CD34, CD38, CD33, c-Kit cell surface expression) co-expressed Flt3Va antigen in 50% samples evaluated. An analysis of Flt3Va protein localization by live cell imaging showed a punctate distribution of Flt3Va on the cell surface. Furthermore, we observed that overexpression of Flt3Va in TF-1 cells led to GM-CSF growth factor independence. Analysis of TF-1 cells in the absence of GM-CSF and Flt3 ligand demonstrated constitutive activation of STAT5, an important mediator of Flt3 signaling, in Flt3Va overexpressing cells. In addition, Erk1/2 phosphorylation was also increased in Flt3Va overexpressing cells, another downstream effector of Flt3. In an effort to determine if Flt3Va+ cells had tumor repopulating ability, we sorted 0.3X10^6 Flt3Va+ and Flt3Va- cells from a PDX sample and injected the sorted populations or unsorted bulk tumor cells into NSG mice. The human cell engraftment in the mice was detected by the expression of human CD45, CD33, CD34, CD38, and c-kit antigens in the peripheral blood. In two experiments, mice injected with Flt3Va+ cells had detectable circulating leukemic cells by ~18 days after injection, while those injected with Flt3Va- cells had detectable circulating leukemic cells after the 4th week. These results suggest both Flt3Va+ and Flt3Va- cell populations are able to reconstitute leukemia after transplantation in NSG mice. However, Flt3Va+ may be expressed by an aggressive AML clone that facilitate early tumor engraftment. Overall, these studies suggest that Flt3Va is a leukemia-specific neoantigen and is an attractive potential immunotherapeutic target in AML. Proteins such as Flt3Va generated by alternative splicing are common in AML and may be targets for of novel blocking antibodies or ADCs, minimizing effects on normal tissues. Disclosures Adamia: Janssen: Research Funding. Nemeth:Janssen: Employment. Attar:Janssen: Employment. Letai:AbbVie: Consultancy, Research Funding; Tetralogic: Consultancy, Research Funding; Astra-Zeneca: Consultancy, Research Funding. Steensma:Millenium/Takeda: Consultancy; Celgene: Consultancy; Amgen: Consultancy; Janssen: Consultancy; Ariad: Equity Ownership; Genoptix: Consultancy. Weinstock:Novartis: Consultancy, Research Funding. DeAngelo:Novartis: Consultancy; Ariad: Consultancy; Pfizer: Consultancy; Baxter: Consultancy; Celgene: Consultancy; Incyte: Consultancy; Amgen: Consultancy. Stone:Agios: Consultancy; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celator: Consultancy; Juno Therapeutics: Consultancy; Roche: Consultancy; Jansen: Consultancy; Pfizer: Consultancy; ONO: Consultancy; Sunesis Pharmaceuticals: Consultancy; Merck: Consultancy; Xenetic Biosciences: Consultancy; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy; Amgen: Consultancy; Karyopharm: Consultancy; Seattle Genetics: Consultancy. Griffin:Janssen: Research Funding; Novartis: Consultancy, Research Funding.

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