Bone Marrow Function Research Articles

FDA-Approved Drug Screening for Compounds That Facilitate Hematopoietic Stem and Progenitor Cells (HSPCs) Expansion in Zebrafish.

Hematopoietic stem cells (HSCs) are a specialized subset of cells with self-renewal and multilineage differentiation potency, which are essential for their function in bone marrow or umbilical cord blood transplantation to treat blood disorders. Expanding the hematopoietic stem and progenitor cells (HSPCs) ex vivo is essential to understand the HSPCs-based therapies potency. Here, we established a screening system in zebrafish by adopting an FDA-approved drug library to identify candidates that could facilitate HSPC expansion. To date, we have screened 171 drugs of 7 categories, including antibacterial, antineoplastic, glucocorticoid, NSAIDS, vitamins, antidepressant, and antipsychotic drugs. We found 21 drugs that contributed to HSPCs expansion, 32 drugs’ administration caused HSPCs diminishment and 118 drugs’ treatment elicited no effect on HSPCs amplification. Among these drugs, we further investigated the vitamin drugs ergocalciferol and panthenol, taking advantage of their acceptability, limited side-effects, and easy delivery. These two drugs, in particular, efficiently expanded the HSPCs pool in a dose-dependent manner. Their application even mitigated the compromised hematopoiesis in an ikzf1−/− mutant. Taken together, our study implied that the larval zebrafish is a suitable model for drug repurposing of effective molecules (especially those already approved for clinical use) that can facilitate HSPCs expansion.

Dopamine signaling regulates hematopoietic stem and progenitor cell function

AbstractHematopoietic stem and progenitor cell (HSPC) function in bone marrow (BM) is controlled by stroma-derived signals, but the identity and interplay of these signals remain incompletely understood. Here, we show that sympathetic nerve–derived dopamine directly controls HSPC behavior through D2 subfamily dopamine receptors. Blockade of dopamine synthesis, as well as pharmacological or genetic inactivation of D2 subfamily dopamine receptors, leads to reduced HSPC frequency, inhibition of proliferation, and low BM transplantation efficiency. Conversely, treatment with a D2-type receptor agonist increases BM regeneration and transplantation efficiency. Mechanistically, dopamine controls expression of the lymphocyte-specific protein tyrosine kinase (Lck), which, in turn, regulates MAPK-mediated signaling triggered by stem cell factor in HSPCs. Our work reveals critical functional roles of dopamine in HSPCs, which may open up new therapeutic options for improved BM transplantation and other conditions requiring the rapid expansion of HSPCs.

TRLS-06. A Phase 1–2 clinical trial of EO1001 (APL-122), a novel irreversible pan-ErbB inhibitor with promising brain penetration

CNS metastases are a prominent driver of cancer morbidity and mortality, especially as targeted therapies have improved systemic outcomes. Mutations in the ErbB/HER kinase family are known oncodrivers in many cancers. Extensive cross-talk among ErbB/HER receptors suggests that inhibition of multiple family members may benefit treatment and limit drug resistance. There is a desperate need for new agents that are more tolerable and effective in treating CNS metastases. EO1001 (APL-122) is a first-in-class, oral, irreversible pan-ErbB inhibitor targeting ErbB1, ErbB2 and ErbB4 with promising CNS penetration in preclinical models. Preclinical data suggests a favorable pharmacokinetic and safety profile and activity against ErbB-driven cancers in patient-derived xenograft models.We report on a first-in-human Phase 1–2 clinical trial in progress. Adult participants with confirmed ErbB-positive cancer, including patients with CNS involvement, who have progressed after standard-of-care, with adequate bone marrow, renal and liver function are eligible.Materials and methods Escalation: One subject per dose cohort is enrolled in an accelerated dose-escalation design until drug-related toxicity (≥G2) is observed in the first cycle, after which dose escalation will revert to a 3 + 3 design to determine the maximum tolerated dose (MTD). Cycle 1: Patients receive a single oral dose of EO1001 on day 1; single-dose pharmacokinetics are measured. Beginning on day 8, EO1001 is administered once daily for 21 days; multi-dose pharmacokinetics are measured. Cycles 2–6: EO1001 is administered once daily in continuous 28-day cycles for up to 20 weeks. Expansion: EO1001 is administered once daily to 20 patients at the MTD in continuous 28-day cycles for up to 6 cycles to determine a recommended Phase 2 dose (RP2D) for further study.Toxicity is assessed based on NCI CTCAEv5 and tumor response is assessed by RECIST 1.1. CNS exposure is evaluated in patients via CSF collection with confirmed CNS disease involvement.

Bone marrow-mesenchymal stem cell-derived exosomal microRNA-141 targets PTEN and activates β-catenin to alleviate myocardial injury in septic mice

Background Mesenchymal stem cells (MSCs) and their derived exosomes have shown potentials in the control of myocardial dysfunction. This study aimed to reveal the function of bone marrow (BM)-MSC-derived exosomes in sepsis-induced myocardial injury and the molecular mechanism. Methods BM-MSC-derived exosomes were obtained and identified. A mouse model with sepsis was induced by cecalligation puncture (CLP) and treated with exosomes. The myocardial function of mice, the production of creatine kinase MB (CK-MB) and lactate dehydrogenase (LDH) in serum, the phosphorylation of a key myocardial contractility-related protein phospholamban (PLB), and the pathological changes in the myocardial tissues were examined. A microRNA (miRNA) microarray analysis was performed to examine the candidate miRNAs carried by the exosomes. Rescue experiments were conducted to validate the involvement of miR-141. Results CLP treatment led to sepsis and notably reduced the myocardial function in mice. Further treatment of BM-MSC-derived exosomes alleviated the CLP-induced myocardial impairment, production of CK-MB and LDH, and inflammatory infiltration and cell apoptosis in mouse myocardial tissues, and restored the PLB phosphorylation. miR-141 was the most upregulated miRNA in the myocardial tissues after exosome treatment. Downregulation of miR-141 blocked the myocardium-protective functions of the exosomes. miR-141 was found to bind to and suppress PTEN expression, which further enhanced the activity of β-catenin. Conclusion This study suggested that BM-MSC derived exosomes ameliorates myocardial injury in septic mice through conveying miRNA-141 and regulating the PTEN/β-catenin axis, and exosomes may serve as promising tools for the management of myocardial injury induced by sepsis or other factors.

Combining Ibrutinib With Rituximab, Cyclophosphamide, Doxorubicin, Vincristine, and Prednisone (R-CHOP): Updated Results From a Phase 1b Study In Treatment-Naïve Patients With CD20-Positive B-Cell Non-Hodgkin's Lymphoma (NHL)

IntroductionR-CHOP is the currently accepted standard of care for a number of the most common B-cell malignancies, including several NHL subtypes. However, despite good initial results observed with R-CHOP, a proportion of patients with NHL either fail to respond or relapse after initial remission. Ibrutinib is a first-in-class, oral, covalent Bruton's tyrosine kinase inhibitor that has demonstrated single-agent activity in a variety of relapsed or refractory B-cell malignancies with limited toxicity (Advani, JCO 2013; Byrd, NEJM 2013; Wang, NEJM 2013), making it an appropriate drug to combine with standard R-CHOP chemotherapy in patients with previously untreated NHL. The primary objective of this phase 1b study was to determine the recommended phase 2 dose (RP2D) of ibrutinib in combination with standard R-CHOP. Secondary objectives were to evaluate safety, the overall response rate (ORR), pharmacokinetics (PK), and pharmacodynamic biomarkers. MethodsThis was an open-label, nonrandomized, multicenter study consisting of 2 parts: dose escalation (Part 1) and dose expansion (Part 2). In Part 1, treatment-naïve patients with histopathologically confirmed CD20-positive B-cell NHL (diffuse large B-cell lymphoma [DLBCL], mantle cell lymphoma, or follicular lymphoma received ibrutinib at 280, 420, or 560 mg/d combined with standard doses of R-CHOP (rituximab, cyclophosphamide, doxorubicin, and vincristine on Day 1, and prednisone on Days 1-5 of each 21-day cycle for up to 6 cycles). Eligibility criteria included stage 1AX to stage IV disease, ≥ 1 measurable disease site, an Eastern Cooperative Oncology Group score of 0-2, and adequate bone marrow, liver, and renal function. Safety was evaluated by monitoring adverse events (AEs) and clinical laboratory evaluations. Patients were evaluated for dose-limiting toxicities (DLTs) during the first 21 days of treatment (Cycle 1). Efficacy assessments were based on serial radiographs and response determined according to the Revised Response Criteria for Malignant Lymphoma at the end of Cycle 3, end of Cycle 6/end of treatment, and at suspicion of progressive disease (Cheson, JCO 2007). In Part 2, eligible patients with newly diagnosed DLBCL received ibrutinib at the RP2D with standard doses of R-CHOP. ResultsA total of 33 patients were enrolled (Part 1, 17; Part 2, 16): male, 51.5%; median age, 61.0 (range 22-81) years. As of end of July 2013, 29 patients (88%) completed 6 therapy cycles, 3 patients (9%) discontinued prematurely (1 study drug noncompliance, 1 non-DLT-related AE, and 1 rituximab reaction before ibrutinib exposure), and 1 patient is ongoing in the last treatment cycle. During Part 1, 2/7 patients in the 280 mg cohort had DLTs: 1 transient syncope and 1 periorbital cellulitis; at 420 mg, 0/4 patients had DLTs; at 560 mg, 1/6 patients had gastritis (grade 2). The RP2D was established as 560 mg ibrutinib. Across Parts 1 and 2, the most common all-grade AEs (≥ 25%) were neutropenia (67%), nausea (67%), thrombocytopenia (61%), vomiting (48%), anemia (36%), fatigue (30%), diarrhea (30%), headache (27%), constipation (27%), and alopecia (27%). AEs ≥ grade 3 were reported in 24 patients; the most common were neutropenia (61%), thrombocytopenia (21%), and anemia (18%). Serious AEs were reported in 15 patients. The current ORR for all evaluable patients across Parts 1 and 2 is 100%: Part 1 (n = 15; final data), complete response (CR) 73%, partial response (PR) 27%; Part 2 (n = 15; interim data), CR 60%, PR 40%; DLBCL patients (n = 22; interim data), CR 64%, PR 36%. Final data for Part 2 and DLBCL patients will be available for ASH. R-CHOP did not affect ibrutinib PK; ibrutinib did not alter vincristine (CYP3A4 substrate) PK. Ibrutinib exposure with R-CHOP was consistent with data from previous single-agent ibrutinib studies. Immunohistochemistry analyses of DLBCL samples from 8 patients subtyped by the Hans algorithm (Hans, Blood 2004) showed 6 patients as GCB subtype and 2 patients as ABC subtype. ConclusionThe combination of ibrutinib and R-CHOP has an acceptable safety profile in treatment-naïve patients with NHL, with no new toxicities noted. The expanded 560 mg ibrutinib + R-CHOP cohort is ongoing to further explore safety and efficacy of the combination in patients with newly diagnosed DLBCL and final analysis is expected by October 2013. Based on the promising early results, a randomized phase 3 trial of R-CHOP +/- ibrutinib is ongoing in de novo DLBCL. Disclosures:Younes:Pharmacyclics, Janssen, Seattle Genetics,Millenium, Incyte : Honoraria. Flinn:J&J/Janssen/Pharmacyclics: Research Funding. Seetharam:Janssen: Employment. Hellemans:Janssen: Employment, Equity Ownership. Smit:J&J: Employment. Badamo-Dotzis:Quintiles, providing services on behalf of Janssen Research & Development, division of Janssen-Cilag, Issy-Les-Moulineaux, France: Employment. Fourneau:J&J: Employment.

An Unbalanced Monocyte Macrophage Polarization in the Bone Marrow Microenvironment of Patients with Poor Graft Function after Allogeneic Hematopoietic Stem Cell Transplantation

BackgroundPoor graft function (PGF) remains a life-threatening complication following allogeneic hematopoietic stem cell transplantation (allo-HSCT), and the underlying mechanisms have not yet been elucidated. Considerable evidence from murine studies has demonstrated that effective hematopoiesis depends on the specific bone marrow (BM) microenvironment, where hematopoietic stem cells (HSCs) reside. In this regard, we previously reported that PGF patients had impaired BM endosteal cells and endothelial progenitor cells, as well as dysregulated T cell responses in the BM microenvironment, which may be involved in the occurrence of PGF (BBMT 2013; BMT2016; Oncotarget 2016; BBMT 2016; Blood 2016; J Transl Med2017). Macrophage is one of the important components of BM immune microenvironment. Meanwhile, Murine studies suggest that macrophages regulate the retention of HSCs by regulating osteoblastic cell activity and nestin-expressing cells. Increasing evidence shows that BM resident macrophages are indispensable for HSCs function and BM erythroid output. However, little is known about the quantity and function of BM macrophages and whether the unbalanced BM macrophages directly interact with HSCs in PGF patients post allo-HSCT.AimsTo compare the number and function of BM macrophages between patients with PGF and GGF after allo-HSCT. Moreover, to investigate whether the unbalanced BM macrophages directly interact with HSCs in PGF patients.MethodsThis prospective nested case-control study enrolled 30 patients with PGF, 60 matched patients with good graft function (GGF), defined as persistent successful engraftment after allotransplant, and 30 healthy donors (HD). Standard monocyte subsets, defined by cluster of differentiation CD14 and CD16, were quantified by flow cytometry. In addition, based on the phenotype of polarized macrophages in vitro, classically activated inflammatory macrophage (M1:CD68+CCR2+) and alternatively activated anti-inflammatory macrophage (M2:CX3CR1+CD163+/CD206+) from BM samples were analyzed by flow cytometry. BM CD14+ monocytes were isolated from BM mononuclear cell (BMMNCs) and were cultured with 100 ng/ml recombinant human M-CSF for 10 days to obtain macrophages. The functions of BM derived macrophages were evaluated by migration assay and phagocytosis assay. CD34+ cells from healthy donors were co-cultured with BM derived macrophages from PGF patients, and the levels of reactive oxygen species (ROS) and apoptosis in CD34+ cells were analyzed by flow cytometry after 5 days co-culture.ResultsAlterations in standard monocyte subsets (classical, intermediate and non-classical) were found when comparing subjects among PGF, GGF and normal controls. In particular, elevated intermediate and non-classical monocyte subsets were observed in PGF patients when compared with those in GGF patients. Moreover, PGF patients displayed an imbalanced M1/M2 ratio compared with the GGF and normal controls, attributable to a reduction in M2 and an increase in M1. Dysfunctional BM derived macrophages, which were characterized by impaired proliferation, migration, phagocytosis and higher level of apoptosis, were revealed in patients with PGF. BM CD34+ cells from healthy donors were co-cultured respectively with BM derived macrophages from patients with PGF and normal controls. Apoptosis and intracellular levels of ROS were markedly increased in CD34+ bone marrow cells which were co-cultured with PGF patients' BM derived macrophages compared with normal controls.Summary/ConclusionIn summary, the current study demonstrated the abnormal number and impaired function of BM macrophages and unbalanced BM macrophages directly interact with HSCs in PGF patients. We speculate that reduction of anti-inflammatory M2 monocytes while increase of pro-inflammatory M1 monocytes in PGF patient BM have a negative impact on HSCs in BM microenvironment. Thus, our results indicate that unbalanced BM M1/M2 and dysfunctional BM macrophages may play an important role in the pathogenesis of PGF following allo-HSCT. Therefore, it would be of value to investigate the appropriate approach to balance the M1/M2 and enhance function of BM macrophages, which may be a promising therapeutic approach for PGF patients. DisclosuresNo relevant conflicts of interest to declare.

Synthetic Lethality Targeting the Aid/RAD51 Axis for Treatment of AICDA-Expressing Lymphoma

Activation Induced Cytidine Deaminase (AICDA or AID) is a DNA directed cytidine deaminase that induces somatic hypermutation and immunoglobulin class switching in stimulated B-lymphocytes. AICDA is a DNA damaging enzyme, producing DNA base pair mismatches that can subsequently be converted into mutations, DNA single strand breaks (SSB), or DNA double strand breaks (DSB). Although AICDA is normally expressed at high levels only in activated B-cells, a wide range of cancer types show constitutive overexpression of AICDA. In malignant cells, as in B-cells, AICDA promotes hypermutation and genomic instability. We previously demonstrated that cells expressing AICDA are critically dependent on the DNA repair factor RAD51 for their survival. We have developed novel small molecule inhibitors of RAD51 that selectively kill AICDA overexpressing cells. One of these, CYT01A, depletes the nucleus of RAD51, thus reducing DNA double strand break repair activity. We have shown CYT01A to be a potent (EC50~150nM) RAD51 inhibitor that reduces the efficiency of RAD51 focus formation, leading to sensitization to excess DNA damage while preserving baseline recombination/repair activity. Here we present the in vitro and in vivo pharmacologic characterization of CYT01A. We first analyzed potential off-target liabilities by carrying out kinome inhibition and safety panel (Panlabs) screens. CYT01A showed negligible inhibitory activity against 429 kinases, receptors, or other pharmacologically important non-RAD51 targets. These data are consistent with high specificity for AICDA and predict in vivo tolerability. We next measured metabolic stability of CYT01A in mouse liver microsome incubation assays, demonstrating CYT01A stability in both the absence and presence of NADPH. Measurement of Caco-2 cell permeability, as a predictor of oral bioavailability, revealed low to moderate efflux of CYT01A. Consistent with these permeability data, CYT01A showed modest oral availability, but relatively long clearance times. Daily oral dosing at 80 mg/kg for 7-14 days resulted in significant reduction in AICDA expressing lymphomas, in a human-to-mouse xenograft model. This demonstrates efficacious iin vivo exposure despite relatively low oral availability. By contrast, AICDA null lymphomas showed no significant tumor response versus vehicle controls, consistent with the synthetic lethal relationship between AICDA and RAD51. Finally, we demonstrated that repeat daily administration of CYT01A up to the maximum dose tested (80mg/kg) was well tolerated and resulted in no observable changes in complete blood counts (CBC), bone marrow function or output, or body weights. Taken together these data represent a definitive proof-of-principle demonstration that in vivo targeting of RAD51 is an effective strategy to treat AICDA expressing cancers. These data form the basis for continued preclinical development of an AICDA/RAD51 synthetic lethal therapeutic paradigm that may be applicable to a wide range of both solid tumors and hematologic malignancies. DisclosuresMills:Cyteir Therapeutics: Employment, Equity Ownership. Cyr:Cyteir Therapeutics: Employment, Equity Ownership. Maclay:Cyteir Therapeutics: Employment, Equity Ownership. Day:Cyteir Therapeutics: Employment, Equity Ownership. Patrick:Cyteir Therapeutics: Consultancy, Equity Ownership. Clas:Cyteir Therapeutics: Consultancy.

Decitabine As Hemoglobin F Inducer in Hemoglobin E/β Thalassemia

IntroductionPhenotypic severity of HbE-beta thalassemia depends on hemoglobin F Level. Gamma globin chain induction has the potential of increasing HbF and decreasing transfusion dependence, and disease severity. Decitabine as a hypomethylating agent can activate repressed gamma globin gene and lead to increase in hemoglobin F.ObjectivesTo study effect of Decitabine on hemoglobin level, hemoglobin F and transfusion requirement as well as its adverse effects.Materials and MethodsThirty patients of HbE Beta thalassemia were enrolled. Both transfusion dependent (TDT) and non-transfusion dependent patients (NTDT) were included after obtaining informed consent. Inclusion criteria included - age greater than 18 years, free of transfusion for at least 90 days before entry (only for NTDT to get better idea of baseline hemoglobin), without any co-morbidity.Participants received 0.2 mg/kg of 5-aza-2′-deoxycytidine (decitabine) subcutaneously on two consecutive days a week for at least 12 weeks. Complete hemogram was done every two weeks and HPLC at every four week interval, till two months after last dose of drug. Patients were transfused packed red blood cells (PRBC) if symptomatic or if Hb < 5gm/dl.ResultsThirty patients (21- NTDT and 9 - TDT), were included in this study. Study comprised of 16 males (53.3 percent) and 14 females (46.7 percent) with a mean age of 29.33 years (summarized in table 1). Hemoglobin increment of > 1gm/dl was seen in 7 (33.3 percent) and >0.5 gm/dl was seen in 8 (38.1 percent) patients of NTDT group. For NTDT group peak mean increment in hemoglobin was 0.938 gm/dl (p value <.001, 95 percent confidence interval 1.33 to 0.539). Detailed results and paired analysis given in table 2. Graph A and Graph B shows trends in hemoglobin and hemoglobin F percentage respectively.Transfusion requirement decreased to 0.24 units compared to 0.96 units per patient per month for TDT patients over a period of last one year. Out of the nine transfusion dependent patients five (55 percent cases) became transfusion independent and continued to remain so during mean follow up period of 6 months (range: 4months to 10 months), three patients continued to remain transfusion independent even after discontinuation of therapy.Decitabine was well tolerated, no hematological toxicity was documented. Regarding non-hematological toxicities, 1 patient complained of chest tightness (grade 1), 1 patient had malena (grade III), endoscopy of this patient showed gastric erosion, 3 patients developed respiratory tract infection (grade I/II- managed on day care basis), 1 patient complained of small joint pain (grade I).DiscussionVarious drugs like hydroxyurea, 5-azacytidine, decitabine, erythropoietin stimulating agents have been evaluated as hemoglobin F inducing agents. Hydroxyurea has shown beneficial effect in HbE Beta Thalassemia patients, with range of response being in between 1% and 90%, and averaging at 20% (Musallam KM et. al Blood 2013). The efficacy of agents such as hydroxyurea is compromised by the suppressive effects on bone marrow function. Hydroxyurea has mutagenic effect also. (Bradai M et al. Transfusion. 2007).Few studies noted a decline in hematological response beyond 12 months (Rigano P et al. BJH 2010). So an alternative is required.Hypomethylating agents can cause activation of gamma globin gene and hence, use of decitabine can be a promising strategy. The only study done previously, showed increased total hemoglobin and absolute fetal hemoglobin. Treatment was well tolerated, with the main adverse event being an elevation in platelet counts (Oliviero NF et al. Blood 2011).In the present study relatively larger number of patients were followed up for a longer period of time and included both TDT and NTDT group.Decitabine was found to increase both hemoglobin and hemoglobin F in NTDT group. In the TDT group it decreased transfusion requirement significantly with increase in hemoglobin F percentage.Even though this study showed good response in HbE/ Beta thalassemia, a randomized control trial with hydroxyurea, with even longer duration of follow up needs to be done to compare both the drugs.ConclusionDecitabine is well tolerated, and is efficacious in increasing hemoglobin F percentage in HbE Beta thalassemia patients. There is decrease in transfusion requirement in TDT group. There is improvement in total hemoglobin as seen in NTDT group. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Recreating the Hematopoietic Microenvironment in Vitro Using Microfluidic “on-a-Chip” Technology Enables the Study of Interactions of Normal and Malignant HSPC with Specific Bone Marrow Niches

Under normal conditions, hematopoietic stem/progenitor cells (HSPC) reside within specific bone marrow (BM) niches. These are comprised of an array of different cell types located strategically to provide a myriad of chemical signals and physical interactions that maintain HSPC and orchestrate the process of hematopoiesis. In human myeloid malignancies, the BM niche is remodeled by malignant cells, which displace resident HSPC, and create self-reinforcing malignant niches that drive disease progression, drug-resistance, and relapse. Attaining a mechanistic understanding of the major determinants of HSPC function in the human BM has been difficult, due to challenges associated with visualizing and modeling the complex niche environment in humans. The creation of accurate models of normal and malignant human BM niches would fill a critical gap, and enable the development of therapies targeting deviant cells and/or signaling pathways, to disrupt self-reinforcing malignant niches and reestablish normal hematopoiesis. We showed that microfluidic “on-a-chip” technologies using bioengineered 3D human tissue constructs, in the presence of physiological flow, can model normal physiology and disease processes and enable drug screening. Herein, we sought to exploit the high physiological relevance and single cell resolution offered by “on-a-chip” technology to recreate the BM microenvironment in vitro and study, in real-time, the interactions of normal and malignant HSPC with each specific niche cell type. To accomplish this objective, we constructed a microfluidic platform using polydimethylsiloxane (PDMS) and photolithography, in which microchannels allow continuous flow of media and HSPC through each of the specific niches, reproducing, in effect, a primitive circulatory system. To recreate the various BM niches, mesenchymal cells (Stro-1+; MSC), arterial endothelium (CD146+NG2+; AEC), and sinusoidal endothelium (CD146+NG2-; SEC) were magnetically-sorted from adult human BM. An aliquot of Stro-1+ cells was also induced to undergo osteogenic differentiation, to generate differentiated osteoblasts (OB). To create the in situ patterned 3D niche-on-a-chip (NOC), the chambers of the microfluidic device were filled with AEC, SEC, MSC, or OB embedded in a hyaluronic acid (HA)-based hydrogel precursor, and the cellularized constructs were formed by UV photopolymerization through the apertures of a photomask. Following flushing of uncrosslinked hydrogel precursor solution, the niche constructs were allowed to equilibrate for 3 days, under constant media flow using a four-channel precision microperistaltic pump. U937, MOLM13, and normal CD34+ cells were each labeled with Qtracker 605, and independently perfused into the main channel of a separate NOC, from which theyenter and evenly disperse into four equidistant chambers, each of which housed a distinct BM niche (AEC, SEC, MSC, or OB). LIVE/DEAD staining confirmed viability of the HSPC and niche cells for > 5 days following HSPC infusion, and immunocytochemistry demonstrated continued expression of appropriate phenotypic markers by each specific niche cell. At 24h post-infusion, U937 cells already exhibited a marked predilection for AEC, and this persisted throughout the 5-day observation period, with roughly 4.5-times more U937 cells engrafting within the AEC niche than the SEC or MSC niches, and 3-times more engrafting within the AEC niche than the OB niche. In contrast, MOLM13 cells exhibited a marked preference for the MSC and SEC niches, a moderate affinity for the OB niche, and engrafted only minimally within the AEC niche. In contrast to malignant cells, normal CD34+ HSPC engrafted preferentially within the SEC and OB niches, exhibited moderate engraftment within the MSC niche, and did not engraft the AEC niche. In conclusion, our studies establish the feasibility of using microfluidic “on-a-chip” technology to recreate the various niches within the BM microenvironment, and provide proof that this novel system can be used to study the interactions of normal and malignant HSPC with distinct cells of the niche. We are currently using this system to delineate the signaling pathways responsible for the observed preferential HSPC/niche cell interactions, with the ultimate goal of using this knowledge to develop more effective treatments for hematological malignancies and enhance engraftment following HSPC transplant. DisclosuresNo relevant conflicts of interest to declare.

Phase 1 Results from ZUMA-6: Axicabtagene Ciloleucel (axi-cel; KTE-C19) in Combination with Atezolizumab for the Treatment of Patients with Refractory Diffuse Large B Cell Lymphoma (DLBCL)

▪Background: Axi-cel, an autologous anti-CD19 chimeric antigen receptor (CAR) T cell therapy, has shown promising efficacy in refractory aggressive non-Hodgkin lymphoma in the ZUMA-1 trial with an objective response rate (ORR) of 82%, including 54% complete responses (CRs; Locke et al. AACR 2017. #9986). Checkpoint expression within the tumor environment, at baseline and post-CAR T cell infusion, suggests that checkpoint blockade could augment axi-cel activity (Galon et al. ASCO 2017. #3025; Vranic et al. PLOS One. 2016). ZUMA-6 is a phase 1/2 study evaluating the safety and efficacy of combination treatment with axi-cel and the anti-PD-L1 antibody, atezolizumab (atezo), in patients with refractory DLBCL (NCT02926833).Methods: Eligible patients (≥ 18 years) had received ≥ 1 prior CD20-targeting and anthracycline-containing regimen with stable or progressive disease to last line of therapy or early relapse after autologous stem cell transplant, had an ECOG ≤ 1, and had adequate bone marrow and organ function. Patients received low-dose conditioning with fludarabine 30 mg/m2/day and cyclophosphamide 500 mg/m2/day × 3 days, followed by axi-cel infusion at a target dose of 2 × 106 cells/kg. In the phase 1 portion of the study, in cohorts 1, 2, and 3, atezo 1200 mg was administered every 21 days for 4 doses starting on day 21, 14, and 1 post-axi-cel infusion, respectively. This report describes phase 1 results from the first 2 dosing cohorts. The primary endpoint of phase 1 was incidence of dose-limiting toxicities (DLTs) within 21 days from the first atezo dose. Secondary endpoints included frequency of adverse events (AEs), ORR, and biomarkers.Results: As of the June 23, 2017 data cut-off, there were 6 patients dosed with axi-cel and atezo (3 in cohort 1; 3 in cohort 2). Median age was 52 years (range, 29-66). Patients had a median of 3 prior therapies (range, 2-4), 4 had disease progression as best response to last prior treatment before study entry, and 3 had an International Prognostic Index score of 3 or 4. No DLTs were observed in either cohort and there was no evidence of worsening or recurrent AEs consistent with cytokine release syndrome (CRS), neurologic events (NE), or other CAR T cell-related toxicities following atezo administration in either dosing schedule. All patients experienced at least 1 AE, the majority of which were cytopenias attributed to conditioning chemotherapy. The most common grade ≥ 3 AEs were anemia (67%), encephalopathy (67%), and hyponatremia (50%). Incidences of grade ≥ 3 CRS and NE were 33% and 67%, respectively. Five patients were evaluable for response; ORR was 100% (5/5) with 1 CR observed. One CR and 2 partial responses are ongoing. All 6 ZUMA-6 patients demonstrated a CAR T cell area under the curve in the first 28 days (AUC28) that was over 2-fold higher than the median observed in patients with DLBCL enrolled in ZUMA-1 (ZUMA-6 range, 816-2301 days × cells/uL; ZUMA-1 median, 374 days × cells/uL). A second peak of > 2-fold over the pre-atezo level in serum concentrations of the immune-modulating cytokine interferon gamma was also observed 1 week after the first atezo infusion in 4/5 evaluable patients. Cohort 3 is currently enrolling and updated results, including those from cohort 3, will be presented.Conclusions: In this ongoing phase 1 study, PD-L1 blockade with atezo following axi-cel infusion appeared to have a manageable safety profile, and translational evidence suggested an enhancing pharmacodynamic action on CAR T cells. Results collected thus far support further evaluation of the combination in the phase 2 portion of the study. DisclosuresLocke:Kite Pharma: Consultancy; Cellular Biomedicine Group Inc: Consultancy. Westin:Novartis Pharmaceuticals Corporation: Membership on an entity's Board of Directors or advisory committees; Apotex: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Kite Pharma: Membership on an entity's Board of Directors or advisory committees. Miklos:Sanofi: Honoraria; Janssen: Consultancy, Honoraria, Other: Travel expenses; Pharmacyclics, LLC: Consultancy, Other: Travel expenses, Patents & Royalties, Research Funding; Kite Pharma: Consultancy, Other: Travel expenses, Research Funding; Adaptive Biotechnologies: Consultancy, Other: Travel expenses; Roche: Research Funding; Genentech: Research Funding; Novartis: Research Funding. Herrara:BMS: Consultancy, Research Funding; Genentech: Consultancy, Research Funding; Merck: Consultancy, Research Funding; Seattle Genetics: Consultancy, Research Funding; Pharmacyclics: Consultancy, Research Funding; Immune Design: Research Funding. Jacobson:Kite Pharma: Consultancy; Pharmacyclics, LLC: Consultancy. Lee:Kite Pharma: Employment, Equity Ownership. Rossi:Kite Pharma: Employment, Equity Ownership. Bot:Kite Pharma: Employment, Equity Ownership. Xue:Kite Pharma: Employment, Equity Ownership. Navale:Kite Pharma: Employment, Equity Ownership. Aycock:Kite Pharma: Employment, Equity Ownership. Wiezorek:Kite Pharma: Employment, Equity Ownership. Roberts:Kite Pharma: Employment, Equity Ownership.

A Phase 1-2 Dose-Escalation and Cohort- Expansion Study of eFT508, a Selective, Orally Bioavailable Inhibitor of MNK1 and MNK2, in Patients with Hematological Malignancies

IntroductionDysregulated translation of messenger RNA (mRNA) plays a role in the pathogenesis of multiple hematological and solid tumors. eFT508, a potent and highly selective small molecule inhibitor of MNK-1 and 2, blocks activation of eIF4E, a key regulator of mRNA translation, and thereby selectively regulates translation of a small set of mRNAs. Significant anti-tumor activity has been observed in preclinical models of diffuse large B-cell lymphoma (DLBCL), particularly in tumor models that have a mutation in MyD88. In addition to direct antitumor activity, eFT508 triggers an anti- tumor immune response and enhances responses to checkpoint inhibitors in preclinical models.The current study is a first-in-human dose escalation and cohort expansion trial of eFT508 in patients with advanced hematological malignanciesMethodsUsing a 3+3 dose escalation schema, cohorts of 3-6 patients (pts) with performance status 0-1, adequate bone marrow, hepatic and renal function and advanced hematological tumors of mainly B cell origin were treated with eFT508 administered orally once daily at a starting dose of 300 mg. A study in pts with solid tumors had previously shown that a dose of 450 mg once daily was well tolerated and based on a pharmacodynamic assay was associated with a degree of target engagement sufficient for maximal efficacy in preclinical models. Efficacy evaluations were performed every 6 weeks.Results6 pts (4 male, 2 female, median age 65 years, range 32-79 years) were treated; 3 at 300 mg daily and 3 at 450 mg daily using an oral suspension of eFT508. 3 pts had DLBCL, 1 had Waldenström macroglobulinemia (WM), 1 had Hodgkin lymphoma (HL) and 1 had follicular lymphoma (FL). The mean number of prior therapies was 4 (range 2-7). Two patients had relapsed after autologous stem cell transplant.The most common adverse events occurring in ≥ 2 pts irrespective of causality, were Grade 1 or 2 unless specified. These included fatigue (n=5), constipation (n=3), hypercalcemia (n=3 with 2 patients experiencing grade 3 toxicity), gastroesophageal reflux (n=2), nausea (n=2) and upper respiratory tract infection (n=2). One of the cases of grade 3 hypercalcemia, felt to be possibly drug related in a patient treated at 450 mg, resolved after a single infusion of zoledronateIn 6 pts evaluated for PK, eFT508 was bioavailable and rapidly absorbed, with a median Tmax of 3 hours and a mean T1/2 of 9.7 hours. Minimal accumulation was observed between Days 1 and 15 in 4 evaluable pts, with a mean accumulation factor of 1.3-fold determined for AUC0-24. Target engagement is being assessed with a pharmacodynamic assay measuring P-eIF4E in peripheral blood cells. In addition, a set of circulating cytokines (including IL-6, IL-8, IL-10, CXCL10 and TNFα) are being measured throughout treatment as MNK1/2 have been shown to regulate cytokine production.Preliminary signs of efficacy were observed with 3 of the 6 pts experiencing stable disease. These included a 32 year old patient with classical HL who had relapsed following multiple lines of chemotherapy, autologous transplant, brentuximab and immune checkpoint inhibition who was treated with 300 mg daily of eFT508. He experienced rapid resolution of B symptoms (fever and night sweats) and at a 6 week scan had a 30% reduction in measurable nodal disease. This nodal reduction was maintained at 12 weeks but a new bone lesion was observedA pt with WM treated at the 450 mg dose level had a reduction in M protein of 22% and remains on treatment for 102 daysA pt with rituximab refractory FL treated at 300 mg remains on treatment with stable disease (6% reduction in tumor volume) for 156 daysConclusionsPreliminary data suggest that eFT508 has acceptable safety and pharmacological activity at the tested dose levels. Dosing has now switched to a capsule formulation that will be used to determine a recommended phase 2 dose. Expansion cohorts for more formal evaluation of efficacy will follow. Updated results will be presented at the ASH meeting. DisclosuresGopal:Seattle Genetics: Consultancy, Research Funding. Ramchandren:Curis: Consultancy; Gilead Sciences: Consultancy; Seattle Genetics: Consultancy; Janssen: Research Funding; Merck: Research Funding; Pharmacyclics: Research Funding. Patel:Gilead: Speakers Bureau; Exelixis: Speakers Bureau; Medivation: Speakers Bureau; Genentech: Speakers Bureau; BMS: Speakers Bureau. Miller:Sunesis Pharmaceuticals, Inc: Consultancy; PRONAI Therapeutics, Inc: Consultancy, Equity Ownership; Presage Biosciences, Inc: Consultancy; FLX Bio, Inc: Consultancy; EpiThany, Inc: Consultancy, Equity Ownership; Acerta Pharma, LLC: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Cleveland BioLabs, Inc: Employment, Equity Ownership; Oncternal Therapeutics, Inc: Consultancy, Equity Ownership; LAM Therapeutics, Inc: Consultancy, Equity Ownership; eFFECTOR Therapeutics, Inc: Consultancy, Equity Ownership; Zucero Pty Ltd: Consultancy. Goel:eFFECTOR THERAPEUTICS: Employment, Equity Ownership. Chiang:eFFECTOR THERAPEUTICS: Employment, Equity Ownership. Webster:eFFECTOR THERAPEUTICS: Employment, Equity Ownership. Sperry:eFFECTOR THERAPEUTICS: Employment, Equity Ownership. Vallner:eFFECTOR THERAPEUTICS: Employment, Equity Ownership. Casseday:eFFECTOR THERAPEUTICS: Employment, Equity Ownership. Barton:eFFECTOR THERAPEUTICS: Employment, Equity Ownership.

Novel Roles of Separase in Hematopoiesis

Separase, an enzyme that resolves sister chromatid cohesion during the metaphase-to-anaphase transition, plays a pivotal role in chromosomal segregation and cell division. In recent years, cancer genome sequencing has revealed several loss of function cohesin mutations in early stages of myeloid, hematologic and epithelial malignancies. These mutations affect the self-renewal and differentiation capacity of HSCs indicating cohesin as a tumor suppressor. Our prior studies indicated that Separase haploinsufficiency acts synergistically with loss of p53 in the initiation and progression of B- and T- cell lymphomas. In contrast to cohesin proteins, the function of the cohesin protease, Separase in hematopoiesis and hematological malignancies has not been well defined.Here we examined the effect of Espl1 heterozygosity using our hypomorphic mouse model (mEspl1 +/hyp ) that has reduced germline Separase activity. The impact of heterozygous loss of mEspl1 allele on total number of HSCs was analyzed by staining with hematopoietic stem and progenitor cells (HSPCs) markers and FACS analysis of cells isolated from bone marrow. Our analysis showed that Separase mutant mice have significantly increased (~2 fold) immunophenotypic HSPCs compared to wild type littermates. To examine whether reduced Separase level elevates self-renewal capacity of HSCs in vivo , we performed competitive transplantation assays to compare differentiation and self-renewal capacity of HSCs in mEspl1+/hyp and WT littermate controls. The outcomes with significantly higher donor derived reconstitution suggested an expansion of HSPCs compartment in mutant mice. In addition, we also observed an altered hematopoietic differentiation with an increase in percentage of donor-derived T- and B-cells in Separase mutant mice. The increased number of HSCs could be a consequence of change in autonomous function of the cells due to reduced level of Separase. To test the cell autonomous function of mEspl1+/hyp , in vitro colony forming unit (CFU) assays were performed in methylocellulose medium. mEspl1+/hyp cells showed significantly higher serial re-plating capacity compared to WT littermate control cells, supporting the notion of cell autonomous function as a probable cause of the observed phenotype.Considering the role of cohesin on gene expression, the effect of heterozygous loss of Separase was analyzed with global transcriptional profiling. Several hematopoietic fingerprint genes like Tle1, Smarca1, Scl2a10, H19, Krt2-8 and Galnt3 were found upregulated. Other genes important in HSC function such as Runx1, Satb1, Hoxa9 and Pbx1 were also upregulated in mEspl1+/hyp mice. The transcriptional profiling identified genes involved in several biological processes such as leukocyte differentiation, cell apoptosis, and chromatin modifications. However, mechanism and direct gene targets of Separase in hematopoiesis or other biological pathways are yet to be identified. To study further on transcriptional regulatory roles and to characterize the direct targets of Separase, ChIP-Seq (cohesin and Separase) and RNA-Seq experiments are being performed in WT and mEspl1+/hyp HSCs.With these studies we hope to better understand the role of cohesin in association with Separase on hematopoiesis particularly the mechanisms that control the fate of HSCs, bone marrow engraftment and function; how cohesin mutations may give rise to malignant transformation, and if this pathway can be targeted for therapeutic intervention. DisclosuresNo relevant conflicts of interest to declare.

Macrophage Involvement in the Response of Acute Myeloid Leukaemia to Chemotherapy

Acute myeloid leukemia (AML) is a clonally heterogeneous neoplasm with poor survival rates. Although chemotherapy often leads to clinical remission, the relapse rate of AML is very high and usually fatal. Chemotherapy is usually effective at debulking AML blasts however a small population capable of re-initiating the leukemia (LIC) survive ultimately causing relapse. Mouse models of AML suggest that LIC are protected from chemotherapy by a protective niche similar to hematopoietic stem cell (HSC) niches. As 1) bone marrow (BM) macrophages are key regulators of HSC niche function and 2) tumor-associated macrophages promote chemotherapy resistance in several models of solid tumors (e.g. breast, lung & ovarian) we investigated a potential role of BM non-malignant macrophages in promoting chemoresistance of AML blasts.Using a mouse model of AML driven by the MLL-AF9 oncogene in which mouse HSC are transduced with MSC-MLL/AF9-ires-GFP and transplanted into syngeneic recipients, we examined how AML progression alters the phenotype of non-malignant GFP-negative BM macrophages by flow cytometry. 18 days post-transplant, GFP+ leukemia burden in the BM was in the 40-60% range. In non-malignant GFPneg BM macrophages, cell surface expression of MHC class II, CD80, L-selectin and neuropilin-1 was significantly decreased suggesting that AML blasts alter the function of BM macrophages.To determine whether BM macrophages could have any role in AML response to chemotherapy, we first determine whether non-malignant BM macrophages were depleted in vivo by treatment with cytarabine (5 days, 100mg/kg) and doxorubicin (3 days, 1mg/kg). Two days after cytarabine+doxoR treatment, most CD11b+ F4/80- Ly6G+ granulocytes and CD11b+ F4/80+ CD169- monocytes were ablated from the BM with 0.1% granulocytes and 1.0 % monocytes surviving after treatment. In contrast CD11b- F4/80+ CD169+ VCAM1+ macrophages were the most resilient myeloid cells in the BM with 9.5% still present 2 days after chemotherapy.To further investigate a possible role of these chemotherapy-resilient CD169+ macrophages in promoting response of AML blasts to chemotherapy, we transplanted MLL-AF9 leukemia cells generated with wild-type HSC into CD169DTR/WT mouse recipients in which one CD169 allele is knocked-in with human diphtheria toxin receptor (DTR). This enables specific depletion of CD169+ macrophages in vivo by injecting diphtheria toxin (DT). In this model, DT treatment targets host CD169+ macrophages but not the AML blasts as they do not contain the DTR knock-in. In the absence of chemotherapy, depletion of recipient CD169+ macrophages did not alter leukemia burden in the BM, spleen or blood 48 hours post depletion and did not alter mouse survival (median survival 29-30 days in both CD169-DTR and WT recipients treated with DT or saline).However when cytarabine + doxoR were administered 48 hours after DT administration, median survival of CD169-DTR recipients was significantly increased to 39.5 days compared to wild-type recipients 35 days, p<0.001. Furthermore leukemia burden in both the blood and spleen was significantly reduced.To determine whether CD169 could by itself mediate lower response of AML to chemotherapy, we transplanted MLL-AF9 cells generated from WT HSC into CD169DTR/DTR mice which are knocked-out for both CD169 alleles. Deletion of both CD169 alleles in recipient mice did not change mouse survival in response to chemotherapy in the absence of DT. Therefore absence of CD169 is not sufficient to prolong mouse survival in response to chemotherapy.In conclusion, non-malignant CD169+ macrophages may protect MLL-AF9 leukemia blasts from the cytotoxic effects of cytarabine and doxorubicin. The mechanisms of this chemoprotection remain to be determined but do not involve a direct effect of CD169 on AML blasts. DisclosuresNo relevant conflicts of interest to declare.

Phase 1 Study of Venetoclax in Combination with Dexamethasone As Targeted Therapy for t(11;14) Relapsed/Refractory Multiple Myeloma

▪Background: Venetoclax (VEN) is a selective, small molecule inhibitor of anti-apoptosis protein BCL-2 that has antitumoral activity against multiple myeloma (MM) cell lines and primary samples, notably those with translocation t(11;14). Recent data suggest that dexamethasone (DEX) enhances the expression of BCL-2 and pro-apoptotic protein Bim, shifting its binding towards BCL-2 and sensitizing MM cells to VEN. Therefore, the combination of VEN with DEX may provide a novel therapeutic approach to improve clinical outcomes in patients with MM, including those with t(11;14) and also those who express higher levels of resistance factors (eg, MCL-1).Methods: This cohort is part of an ongoing, dose-escalation, open-label, phase 1 study (NCT01794520) and it is being conducted to evaluate safety profile, pharmacokinetics (PK) and preliminary efficacy of VEN in combination with DEX in patients with t(11;14)-positive MM. Eligible patients were adults (≥18 years of age) who had previous treatment with a proteasome inhibitor and an immunomodulatory drug, an Eastern Cooperative Oncology Group score ≤1, measurable disease, and adequate bone marrow function. Presence of t(11;14) was confirmed for all eligible patients by means of FISH performed at a central laboratory. Patients were given oral VEN 800 mg/day plus oral DEX 40 mg (20 mg for patients ≥75 years of age) on days 1, 8, and 15 of each 21-day cycle. Preliminary efficacy was assessed as the overall response rate (ORR), time to progression (TTP), and duration of response (DOR) of VEN combined with DEX based on the 2011 International Uniform Response Criteria for Multiple Myeloma.Results: Twenty patients with t(11;14) R/R MM were enrolled (17 [85%] male; median age, 63 years [range, 46-77 years]). International Staging System status was I in 9 patients (45%), II in 7 (35%), III in 3 (15%), and missing in 1 (5%).The median number of prior therapies was 3 (range, 1-7). Sixteen (80%) patients had received bortezomib (11 [55%] refractory), 18 (90%) had received lenalidomide (17 [85%] refractory), and 17 (85%) had prior autologous stem cell transplantation. In addition, 8 patients (40%) were refractory to pomalidomide, and 4 (20%) were refractory to carfilzomib. Common adverse events (AEs) in ≥20% of patients were nausea, diarrhea, or insomnia (35% each); hyperglycemia or hypophosphatemia (30% each); and thrombocytopenia, leukopenia, pyrexia, upper respiratory tract infection, cough, nasal congestion, increased lactate dehydrogenase, increased creatinine, or hypokalemia (20% each). Grade 3/4 AEs in ≥10% of patients were lymphopenia or hypophosphatemia (15% each), as well as hyperuricemia or laboratory tumor lysis syndrome (10% each). Serious AEs in ≥2 patients were 2 cases of laboratory tumor lysis syndrome (10%). No deaths have occurred. ORR (partial response or better) was observed in 65% of patients (13/20) and included 7 very good PRs and 6 PRs. Six discontinuations of study drug occurred, and all were due to disease progression. ORRs were 82% (9/11) and 71% (12/17) in patients refractory to bortezomib and to lenalidomide, respectively. The ORR was also comparable between patients who had 1-3 prior lines of therapy (64% [7/11]) and those who had ≥3 lines (78% [7/9]). The median time to first response was 1.4 months (95% CI, 1.3-2.0 months). As of May 15, 2017, the median time on study was 6 months (range, <1 to 9 months). For this data cut, the median TTP and DOR have not been reached yet, but the estimated 6-month TTP rate was 64% (95% CI, 39-81%).Conclusions: VEN plus DEX demonstrated a tolerable safety profile in patients with t(11;14) R/R MM. Similar to previously reported results with VEN monotherapy, this combination appears to be clinically active regimen in patients whose disease is positive for t(11;14), with 65% of patients achieving a response of PR or better. These early findings support the further investigation of VEN combined with DEX in this patient population. DisclosuresKaufman:Amgen, Novartis: Research Funding; Amgen, Roche, BMS, Seattle Genetics, Sutro Biopharma, Pharmacyclics: Consultancy. Gasparetto:Janssen, BMS, Celgene: Other: Travel, accommodations, or other expenses paid or reimbursed; Celgene: Research Funding; Janssen, BMS, Celgene: Consultancy; Janssen, BMS, Celgene, Takeda: Honoraria. Mikhael:Onyx, Celgene, Sanofi, AbbVie: Research Funding. Moreau:Millennium: Consultancy, Honoraria; Celgene, Janssen, Takeda, Novartis, Amgen, Roche: Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria; Novartis: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Bristol-Myers Squibb: Honoraria; Amgen: Honoraria; Celgene: Consultancy, Honoraria; Onyx Pharmaceutical: Consultancy, Honoraria. Touzeau:AbbVie: Research Funding. Vij:Takeda, Onyx: Research Funding; Celgene, Onyx, Takeda, Novartis, BMS, Sanofi, Janssen, Merck: Consultancy. Facon:Amgen, Celgene: Speakers Bureau. Pegourie:Takeda, Novartis, Janssen, BMS: Consultancy. Benboubker:Takeda, Celgene, Janssen, Amgen: Consultancy. Boise:Abbvie: Consultancy; Eli Lilly and Company: Research Funding. Alzate:AbbVie: Employment, Equity Ownership. Dunbar:AbbVie: Employment, Equity Ownership. Jia:AbbVie: Employment, Equity Ownership. Freise:AbbVie: Employment, Equity Ownership. Verdugo:AbbVie: Employment, Equity Ownership. Ross:AbbVie: Employment, Equity Ownership. Maciag:AbbVie: Employment, Equity Ownership. Kumar:Celgene, Millennium/Takeda, Onyx, AbbVie, Janssen, Sanofi, Novartis, Amgen, Genentech, Merck, Oncopeptides, Roche, Skyline Diagnostics: Research Funding; Skyline: Honoraria; Celgene, Millennium, BMS, Onyx, Janssen, Noxxon, AbbVie, Amgen, Merck, Oncopeptides, Skyline Diagnostics, Takeda: Consultancy.

Altered HSC Programming Drives Aberrant Blood Output Underlying RA Pathogenesis

Rheumatoid arthritis (RA) is a debilitating autoimmune disorder characterized by autoantibody production towards collagen type-II in the synovial joints of the skeletal system, leading to progressive joint damage. RA is accompanied by chronic systemic production of pro-inflammatory cytokines including IL-6, TNF, and IL-1. Importantly, RA patients often suffer from significant hematological complications including production of tissue-damaging macrophages, chronic anemia, impaired lymphopoiesis and suppressed bone marrow function. As blood-forming hematopoietic stem cells (HSC) constitute the root of the immune system, identifying mechanisms by which altered HSC function may contribute to disease could provide key insights into RA pathogenesis and therapeutic intervention.In this study, we used a mouse model of collagen-induced arthritis (CIA), which faithfully recapitulates many features of human autoimmune arthritis, to identify the impact of disease on the hematopoietic compartment, and identify mechanisms underlying alteration in blood lineage output and function. Strikingly, bone marrow hematopoietic populations were significantly altered in CIA mice. This was typified by decreased numbers of peripheral red blood cells (RBC) and bone marrow erythroid progenitors, alongside decreases in bone marrow B cells and common lymphoid progenitors (CLP). On the other hand, we observed increased numbers of platelets, bone marrow granulocytes, and myeloid-biased multipotent progenitors (MPP3). Strikingly, we also observed reductions in the stromal cells comprising the bone marrow niche, with decreases in epithelial, mesenchymal, and osteoblastic cell types, suggesting that the inflammatory effects of RA influence the bone marrow compartment at multiple levels. We functionally validated our phenotypic analyses using colony forming unit (CFU) assays, and observed increased myeloid CFU, with concomitant decreases in erythroid and lymphoid colony forming activity in unfractionated bone marrow cells from CIA mice. Despite these changes, transplantation assays using purified HSCs from CIA and control mice showed that long-term reconstitution potential of HSCs was largely unaltered, suggesting that HSC self-renewal capacity is not significantly impaired. Taken together, these observations suggest that autoimmune arthritis induces substantial remodeling of the bone marrow, resulting in overproduction of myeloid cells at the expense of other lineages.In line with our phenotypic and functional analyses, RNA-seq analysis of HSCs from CIA versus control mice uncovered precocious activation of myeloid-specific gene programs that likely underlie shifts in lineage output. Moreover, in line with our transplantation data, we did not observe significant shifts in the expression of core genes governing HSC self-renewal. Collectively, these data show that the inflammatory environment of RA causes changes to hematopoietic output, leading to an increase in damaging myeloid cell types, further fueling the inflammatory environment and disease progression.We are currently identifying the mechanism underlying myeloid overproduction in the bone marrow, with focus on systemic inflammatory factors and/or shifts in the gastrointestinal microbiota, as potential underwriters of deregulated hematopoiesis and disease development. We are also assessing the functionality of myeloid cells generated by HSCs during RA to determine whether they exhibit a stronger pro-inflammatory phenotype. Ultimately, these studies will increase our understanding of the crosstalk between RA and the hematopoietic system, and potentially opens new avenues for therapeutic intervention to re-establish the hematopoietic system and even rescue RA progression. DisclosuresNo relevant conflicts of interest to declare.

Multi-Institutional Retrospective Analysis of Carbon-Ion Radiotherapy for Patients with Locally Advanced Adenocarcinoma of the Uterine Cervix.

The clinical significance of carbon-ion radiotherapy (CIRT) for adenocarcinoma (AC) of the uterine cervix has been assessed in several single-institutional studies. To validate the significance, we conducted a multi-institutional survey of CIRT for locally advanced AC (LAAC) of the uterine cervix. We retrospectively analyzed the clinical outcomes of patients with stage IIB-IVA LAAC of the uterine cervix who underwent chemo-CIRT or CIRT alone between April 2010 and April 2016. Patients received 74.4 Gy (relative biological effectiveness [RBE]) in 20 fractions of CIRT or 55.2 Gy (RBE) in 16 fractions of CIRT plus three sessions of brachytherapy. Patients aged ≤ 70 years with adequate bone marrow and organ function were administered cisplatin weekly (40 mg/m2 per week for up to 5 weeks). Fifty-five patients were enrolled in this study. The median follow-up period was 67.5 months. The 5-year overall survival (OS) and local control (LC) rates were 68.6% and 65.2%, respectively. Multivariate analysis showed that the initial tumor response within 6 months was significantly associated with LC and OS. The present study represents promising outcomes of CIRT or chemo-CIRT for LAAC of the uterine cervix, especially in the cases showing initial rapid regression of the tumor.

Survival benefit of induction chemotherapy for locally advanced nasopharyngeal carcinoma: prognosis based on a new risk estimation model

BackgroundAlthough the National Comprehensive Cancer Network (NCCN) Guidelines recommend CCRT+AC and IC + CCRT as level 2A evidence for treatment of the locoregionally advanced NPC (II-IVa), IC + CCRT+AC could also be an alternative but it is seldom used because of the low completion rates. This article aimed to compare the effectiveness of the three radiotherapy regimens using a large-scale retrospective study.MethodsThis retrospective single center analysis enrolled 1812 diagnosed NPC patients at Nanfang Hospital from January 2005 to December 2015 and only 729 patients met the inclusion criteria and were analyzed. Patients without distant metastasis, age of 18–70 years, Karnofsky scores of at least 70,stage III-IVb, and adequate adequate bone marrow, liver and renal function. Were enrolled. Adverse events and other categorical variables were compared by Pearson chi-square test or Fishier exact test. Time-to-event data were described with the Kaplan-Meier curves, time-to-event intervals compared with the log-rank test. We did multivariable analyses with the Cox proportional hazards model to test the independent signifi cance of diff erent factors. Cox proportional hazards model was used to estimate the β regression coeffi cient, p value, and hazard ratio and its 95% CI for each of the selected risk predictors.ResultsThe median follow-up time was 47 months. Kaplan-Meier analyses revealed no significant differences among three groups in 3-year failure-free survival (FFS, P = 0.225), 3-year overall survival (OS, P = 0.992), 3-year locoregional failure-free survival (LFFS, P = 0.549), and 3-year distant failure-free survival (DFFS, P = 0.174). Stratified survival analysis based on the risk scoring model revealed no differences in FFS, OS, LFFS, and DFFS between IC + CCRT and CCRT+AC groups for low-risk patients, however, the 3-year OS (88.3% vs. 77.6%, P = 0.049) and 3-year DFFS (84.0% vs.66.8%, P = 0.032) were respectively significantly better in IC + CCRT group compared with CCRT+AC group for high-risk patients.ConclusionsCompared with CCRT+AC, IC + CCRT lowers distant metastasis rate and improves OS among patients with locally advanced NPC in high risk group.

Coastal: A phase 3 study of the PI3Kδ inhibitor zandelisib with rituximab (R) versus immunochemotherapy in patients with relapsed indolent non-Hodgkin’s lymphoma (iNHL).

TPS7573 Background: Patients (pts) with iNHL treated with front-line immunochemotherapy may benefit from an alternative, chemotherapy-free regimen at relapse. Zandelisib, a potent, selective, and structurally differentiated oral PI3Kδ inhibitor, achieved an 87% response rate, with median duration of response not reached in iNHL when given as a monotherapy or in combination with R. A low rate ( &lt; 10%) of Grade ≥ 3 immune-mediated adverse events of special interest associated with PI3kδ inhibitors is observed in patients administered zandelisib on an intermittent schedule (IS) (JCO 2020 38:15_suppl, 8016). An open-label, phase 2 study (TIDAL, NCT03768505) of zandelisib as monotherapy is ongoing in pts with relapsed/refractory follicular lymphoma (FL) and marginal zone lymphoma (MZL). Methods: The COASTAL study is a randomized, open-label, controlled multicenter phase 3 trial to investigate the safety and efficacy of zandelisib in combination with R versus standard immunochemotherapy in pts with iNHL. Key eligibility criteria: adults with relapsed or refractory FL or MZL who received ≥1 prior lines of therapy which must have included an anti-CD20 antibody in combination with chemotherapy or lenalidomide (L); at least one bi-dimensionally measured lesion &gt; 1.5 cm; adequate bone marrow, renal and hepatic function; ECOG performance status score of 0 to 1. Key exclusion criteria: histologically confirmed diagnosis of FL grade 3b or transformed disease; administration of 2 prior immunochemotherapy regimens; prior PI3K inhibitor therapy; known lymphomatous involvement of the central nervous system. Subjects will be randomized 1:1 to receive R-zandelisib or immunochemotherapy (R-CHOP or R-B) and stratified by type and number of prior treatment regimens, histology, and duration of treatment-free interval after last therapy. Zandelisib will be given in a 28-day cycle comprising of daily dosing for 2 cycles followed by IS dosing on days 1-7 of each 28-day subsequent cycle for a duration of 2 years. Rituximab or immunochemotherapy will be given for a total of 6 cycles. Disease response will be assessed by an Independent Response Review Committee according to the modified Lugano Classification. Radiographic tumor assessment will be performed approximately every 12 weeks for the first 9 months, every 16 weeks for the next 12 months, and every 24 weeks thereafter. The primary efficacy endpoint is progression-free survival. The major secondary endpoints include ORR, complete response rate, overall survival, and safety. The trial will enroll approximately 534 pts in ̃200 sites globally and will begin enrollment in mid-2021. Clinical trial information: NCT04745832.

Initial results of the combination of PI3Kδ inhibitor zandelisib (ME-401) and the BTK inhibitor zanubrutinib in patients (pts) with relapsed or refractory (R/R) B-cell malignancies.

7553 Background: Dual inhibition of PI3Kδ and BTK pathways may overcome existing or acquired monotherapy resistance. Dual inhibition of these pathways displays synergistic activity in cell lines that is evident even at suboptimal concentrations [Blood 2015;125(14):2306-09]. Zandelisib is a potent, selective, and structurally differentiated oral PI3Kδ inhibitor (i), and zanubrutinib is an oral BTKi. Based on their efficacy as monotherapy, we hypothesized that the combination of zandelisib and zanubrutinib can be well tolerated and may improve the depth and durability of responses. We evaluatedthis combination therapy in pts with R/R B-cell malignancies to determine the optimal dose and schedule for further evaluation in disease-specific expansion cohorts (NCT02914938). Methods: This is a multi-cohort Phase 1b study enrolling pts with FL, CLL, MZL, MCL, DLBCL, or high grade B-cell lymphoma (HGBCL), ≥1 prior therapy, adequate bone marrow and organ function, ECOG performance status ≤2, and no prior PI3Ki or BTKi therapy. For this combination therapy, two dose levels were evaluated in 28-day cycles: Cohort 10A: zandelisib 60 mg once daily for 2 cycles followed by an intermittent schedule (IS) on days 1-7 of subsequent 28-day cycles and zanubrutinib 160 mg twice daily (bid). Cohort 10C: zandelisib 60 mg on days 1-7 starting in Cycle 1 and zanubrutinib at 80 mg bid. Dose limiting toxicity (DLT) observation period was 28 days for cohort 10A and extended to 56 days for cohort 10C. Response was assessed at month 3, 7, 13 and then every 6 months until progression. Results: 20 pts treated, 7 in cohort 10A and 13 in cohort 10C: 8 FL, 5 CLL, 2 DLBCL, 2 HGBCL, 2 MZL, and 1 MCL. Median age 70 years (range, 44-85) and median prior therapies 2 (1-8). Median follow-up of 2.9 months (0.5-17.4+). There were no DLT in cohort 10A, grade (Gr) ≥3 adverse events (AE) occurred after day 28 in 4 pts, including Gr 4 neutropenia (1 pt), Gr 3 neutropenia, fatigue and CMV colitis (1 pt), Gr 3 AST/ALT and rash (1 pt) and Gr 3 AST/ALT (1 pt). In cohort 10C, 2 pts had DLT with Gr 3 AST/ALT in Cycle 2, with 1 pt successfully resuming both drugs and 1 discontinued treatment due to recurrence of Gr 3 AST/ALT upon rechallenge.. Other Gr 3 AE were all laboratory findings: 1 pt (CLL) had laboratory TLS, neutropenia and thrombocytopenia and 2 pts (FL, DLBCL) had neutropenia. Response rate was 100% (2 CR 14 PR) in the following 16 pts with indolent NHL and MCL evaluable for response: FL (2 CR, 6 PR), CLL (5 PR), MCL (1 PR) and MZL (2 PR). No pt with aggressive B-cell lymphomas has responded. Conclusions: The combination of zandelisib 60 mg on IS from Cycle 1 and zanubrutinib 80 mg bid is well tolerated and achieves a high ORR in R/R indolent B-cell malignancies. This schedule is being evaluated in expansion cohorts in R/R FL and MCL. Clinical trial information: NCT02914938.

Encorafenib and binimetinib with or without nivolumab in treating patients with metastatic radioiodine refractory BRAF V600 mutant thyroid cancer.

TPS6085 Background: Differentiated thyroid cancer is the most common endocrine malignancy and has a high frequency of actionable molecular aberrations including BRAF V600E mutations (45%), RET fusions (10%), and NTRK fusions ( &lt; 2%). FDA approved systemic therapies for metastatic radioiodine refractory differentiated thyroid cancer (RR-DTC) include multikinase inhibitors (Lenvatinib and sorafenib), NTRK inhibitors (larotrectinib and entrectinib for NTRK fusion+ cancers), and RET inhibitors (selpercatinib and pralsetinib for RET fusion+ cancers). Previous phase II clinical trials showed clinical efficacy with first and second generation BRAF inhibitors in patients with BRAF mutant RR-DTC. BRAF inhibitors have not yet been FDA approved for treatment of BRAF mutant RR-DTC. Effective therapeutic options for patients with BRAF mutant RR-DTC remains an important unmet clinical need. BRAF mutant thyroid cancers often show elevated expression of PD-L1. Additionally, BRAF inhibition results in increased expression of PD-L1 in thyroid cancer. This clinical trial seeks to evaluate the safety and efficacy of encorafenib plus binimetinib with or without nivolumab in patients with BRAF mutant metastatic RR-DTC. Encorafenib and binimetinib are highly selective and potent oral inhibitors of BRAF and MEK, respectively. Nivolumab is a potent inhibitor of the immune co-inhibitory receptor programmed cell death protein 1 (PD-1). Methods: This is a phase II, single institution, open-label, randomized clinical trial evaluating the combinations of (Arm 1) encorafenib 450 mg/day + binimetinib 45 mg twice daily and (Arm 2) encorafenib 450 mg/day + binimetinib 45 mg twice daily + nivolumab 480 mg I.V. every 4 weeks in patients with metastatic BRAF mutant RR-DTC. The trial will enroll 20 patients in each arm and treatment will be given in 28 day cycles for up to 2 years. Eligible patients must have metastatic/unresectable BRAF mutant RR-DTC, an ECOG performance status of 0-1 and adequate bone marrow, liver and kidney function. Patients with CNS metastases are included if the metastases have been treated and remained stable or are asymptomatic and ≤10 mm in diameter. Patients may be systemic therapy naïve or have previously been treated with multikinase inhibitors. Prior therapy with BRAF, MEK or immune checkpoint inhibitors is exclusionary. The primary endpoint is confirmed objective response rate (ORR) determined by RECIST v1.1 with restaging imaging every 12 weeks. Secondary endpoints include progression free survival, overall survival, and safety/tolerability (CTCAE v5.0). Arms 1 and 2 will be evaluated independently and are not powered for direct comparison. The trial design includes continuous toxicity monitoring with a Pocock-type stopping boundary. This clinical trial is in progress and 3 patients have been enrolled. Clinical trial information: NCT04061980.