Myelofibrosis (MF) is characterized by progressive bone marrow (BM) fibrosis resulting from aberrant megakaryopoeisis and expression of pro-inflammatory cytokines. These two processes, heavily influenced by bromodomain and extraterminal domain (BET)-mediated gene regulation, lead to myeloproliferation and cytopenias. CPI-0610 is a potent, selective and unique BET inhibitor under investigation in MF patients as monotherapy or in combination with ruxolitinib in the MANIFEST trial (NCT02158858). In ruxolitinib naïve and experienced MF patients, treatment with CPI-0610 monotherapy or in combination with ruxolitinib not only reduced spleen volume and symptoms, but also improved hemoglobin levels and overcame transfusion dependency in a subset of patients. To evaluate the effects of CPI-0610 on BM biology, correlative analyses were conducted using patient samples collected from the MANIFEST trial.
Pre-treatment and post-treatment BM biopsy samples were available from 78 evaluable patients, with reticulin staining conducted and evaluated by local pathologists available for 34 pre-treatment and 24-week post-treatment pairs of BM biopsy samples. Improvement in reticulin staining of at least one grade was observed in 32% (11/34) of patients. Central pathology review of reticulin staining is ongoing and will be presented.
Additional bone marrow biopsy pairs collected pre-treatment and 24-week post-treatment for exploratory histopathological assessments were available from a total of 23 unselected MANIFEST patients at the time of the abstract submission. To assess changes in erythroid and megakaryocytic lineages, H&E and immunohistochemistry (IHC) staining of CD71 and CD61 were conducted centrally. Semi-quantitative analysis revealed an overt increase in erythroid lineage formation in 61% (14/23) of patients. Patients with CD71 improvement in BM IHC generally showed a greater increase in median hemoglobin levels than those without improvement. Tight clusters of megakaryocytes (MK), characteristic in MF BM, were observed at baseline in all cases and were decreased/absent after treatment in 52% (12/23) patients. In addition, decreased MK number to <5/high powered field was also observed in 39% (9/23) of patients. Overall improvement in MK histotopography was observed in 65% of patients (15/23). Central histopathology review with IHC staining, including reticulin staining, will be conducted on additional samples and will be available for presentation at the conference.
To further dissect the underlying mechanism of these relative improvements in bone marrow composition and histotopography induced by CPI-0610, CD34+ hematopoietic stem cells were isolated from peripheral blood collected from multiple MF patients at baseline to evaluate the impact of CPI-0610 on MK and erythroid differentiation in vitro. When CD34+ cells from MF patients were treated with CPI-0610 in erythroid differentiation conditions in the presence of SCF, IL3 and EPO, a dose-dependent increase in more mature erythroid cell populations was observed. However, under the same conditions, ruxolitinib suppressed erythroid differentiation, which is consistent with ruxolitinib inhibition of EPO signaling and the anemia caused by ruxolitinib treatment in MF patients. Suppressive effects of ruxolitinib on erythroid differentiation were partially rescued by CPI-0610 in a dose-dependent manner. CPI-0610 treatment of CD34+ cells from MF patients in MK differentiating conditions in the presence of SCF, IL6, IL9 and TPO resulted in a dose-dependent decrease in proliferation of CD34+ cells and an increase in the ratio of late MK (CD34-/CD41a+/CD42b+) and early MK (CD34+/CD41a+/CD42b+). This CPI-0610-induced dose-dependent maturation of aberrant immature MK cells may play a role in reducing MF disease manifestations. Molecular studies to further our understanding of the underlying mechanisms of CPI-0610 treatment are ongoing.
Taken together, these paired BM biopsy and in vitro myeloid maturation results demonstrated an effect of CPI-0610 in promoting erythroid and MK differentiation. These results may partially explain CPI-0610's clinical effects in MF patients, including rising hemoglobin, reduced transfusion dependency and reduction in spleen volume and symptoms.
Mertz:Constellation Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Keller:Constellation Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Meyer:Constellation Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Zavidij:Constellation Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Cui:Constellation Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Verstovsek:Roche: Research Funding; Gilead: Research Funding; Promedior: Research Funding; Sierra Oncology: Consultancy, Research Funding; Protagonist Therapeutics: Research Funding; AstraZeneca: Research Funding; Genentech: Research Funding; Novartis: Consultancy, Research Funding; Blueprint Medicines Corp: Research Funding; Celgene: Consultancy, Research Funding; CTI Biopharma Corp: Research Funding; PharmaEssentia: Research Funding; Incyte Corporation: Consultancy, Research Funding; ItalPharma: Research Funding; NS Pharma: Research Funding. Gupta:Sierra Oncology: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bristol MyersSquibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy; Incyte: Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Talpaz:IMAGO: Consultancy; Takeda: Research Funding; BMS: Membership on an entity's Board of Directors or advisory committees; Constellation Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Novartis: Research Funding. Harrison:Promedior: Honoraria; AOP Orphan Pharmaceuticals: Honoraria; Roche: Honoraria; Incyte Corporation: Speakers Bureau; Gilead Sciences: Honoraria, Speakers Bureau; CTI Biopharma Corp: Honoraria, Speakers Bureau; Shire: Honoraria, Speakers Bureau; Janssen: Speakers Bureau; Novartis: Honoraria, Research Funding, Speakers Bureau; Celgene: Honoraria, Research Funding, Speakers Bureau; Sierra Oncology: Honoraria. Mead:CTI: Consultancy; Gilead: Consultancy; Novartis: Consultancy, Honoraria, Other: travel, accommodations, expenses, Research Funding, Speakers Bureau; Celgene/BMS: Consultancy, Honoraria, Other: travel, accommodations, expenses, Research Funding; Abbvie: Consultancy. Mesa:Novartis: Consultancy; CTI BioPharma: Research Funding; Bristol Myers Squibb: Research Funding; Sierra Oncology: Consultancy; Genentech: Research Funding; Samus Therapeutics: Research Funding; LaJolla Pharmaceutical Company: Consultancy; Incyte: Research Funding; Promedior: Research Funding; AbbVie: Research Funding. 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). Senderowicz:Constellation Pharmaceuticals: Consultancy, Current equity holder in publicly-traded company, Ended employment in the past 24 months; Puma Biotechnology: Membership on an entity's Board of Directors or advisory committees. Colak:Constellation Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Shao:Constellation Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Luptakova:Constellation Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Humphrey:Constellation Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Wang:Constellation Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Trojer:Constellation Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company.
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