ALBERT

Description: Chronic granulomatous disease (CGD) can result from any of four single gene defects involving the components of the superoxide (O−2 ) generating phagocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. We show that transduction of peripheral blood CD34+ hematopoietic progenitors from a p67phox deficient CGD patient with replication defective amphotropic retrovirus encoding p67phox (MFGS-p67phox) significantly corrected the CGD functional defect in phagocyte oxidase activity in vitro. Using a chemiluminescence assay of oxidase activity, we showed that transduced patient CD34+ progenitors differentiating to myeloid cells in culture produced 25% of the total superoxide produced by normal CD34+ progenitors differentiating in culture. A flow cytometric assay of oxidase activity used to assess the oxidase function of individual cells in the cultures indicated that up to 32% of maturing granulocytes derived from transduced CD34+ progenitors from the p67phox CGD patient were oxidase positive with the average level of correction per granulocyte of 85% of that seen with granulocytes in similar cultures of CD34+ progenitors from normal volunteers. Nitroblue tetrazolium dye reduction assays of colonies of transduced progenitors in soft agar indicated that in some studies restoration of oxidase activity occurred in myeloid cells within 44% of granulocyte-erythrocyte-monocyte colonies, and within 28% of the combined group of granulocyte colonies/monocyte colonies/granulocyte-monocyte colonies. These high correction rates were achieved without any selective regimen to enrich for transduced cells. This study provides a basis for development of gene therapy for the p67phox deficient form of CGD.

Description: Chronic granulomatous disease (CGD) can result from any of four single gene defects involving the components of the superoxide (O−2 ) generating phagocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. We show that transduction of peripheral blood CD34+ hematopoietic progenitors from a p67phox deficient CGD patient with replication defective amphotropic retrovirus encoding p67phox (MFGS-p67phox) significantly corrected the CGD functional defect in phagocyte oxidase activity in vitro. Using a chemiluminescence assay of oxidase activity, we showed that transduced patient CD34+ progenitors differentiating to myeloid cells in culture produced 25% of the total superoxide produced by normal CD34+ progenitors differentiating in culture. A flow cytometric assay of oxidase activity used to assess the oxidase function of individual cells in the cultures indicated that up to 32% of maturing granulocytes derived from transduced CD34+ progenitors from the p67phox CGD patient were oxidase positive with the average level of correction per granulocyte of 85% of that seen with granulocytes in similar cultures of CD34+ progenitors from normal volunteers. Nitroblue tetrazolium dye reduction assays of colonies of transduced progenitors in soft agar indicated that in some studies restoration of oxidase activity occurred in myeloid cells within 44% of granulocyte-erythrocyte-monocyte colonies, and within 28% of the combined group of granulocyte colonies/monocyte colonies/granulocyte-monocyte colonies. These high correction rates were achieved without any selective regimen to enrich for transduced cells. This study provides a basis for development of gene therapy for the p67phox deficient form of CGD.

Description: Background: Polycythemia vera (PV) is a myeloproliferative neoplasm (MPN) characterized by clonal stem cell proliferation of the erythroid, myeloid, and megakaryocytic lines. The predominant clinical characteristic is an increase in red cell mass, resulting in hyperviscosity of the blood, which is responsible for most symptoms during early stages of disease. Disease progression typically results in symptomatic splenomegaly and severe constitutional symptoms, causing significant morbidity and a shortened life expectancy. Patients with PV may develop cardiovascular complications, myelofibrosis (MF), myelodysplasia, or acute myeloid leukemia (AML). Long-term (20-year) survival for PV is 18%, highlighting the poor long-term prognosis and need for additional therapies. Phlebotomy and low-dose aspirin are the standard of care for initial treatment; hydroxyurea (HU) remains the myelosuppressive agent of choice, despite the increased potential for leukemic transformation, estimated at 10% after 13 years of exposure. The Janus kinase (JAK) inhibitor ruxolitinib is approved in the US and Europe for the treatment of patients who have had an inadequate response to or are intolerant of HU. In clinical trials, ruxolitinib produced responses in 23% of patients, compared with

Description: Background: Myelofibrosis (MF) is a myeloproliferative neoplasm (MPN) characterized by progressive bone marrow fibrosis, ineffective erythropoiesis, dysplastic megakaryocyte hyperplasia, and extramedullary hematopoiesis. MF includes primary MF (PMF), post-polycythemia vera MF (post-PV-MF), and post-essential thrombocythemia MF (post-ET-MF). Clinical presentation is heterogeneous, marked by splenomegaly, progressive anemia, and constitutional symptoms. The median survival in patients with high-risk disease is approximately 2 years. Hematopoietic Stem Cell Transplant (HSCT) is a potentially curative therapy, however due to considerable morbidity and mortality rates, HSCT is not appropriate for most patients, including elderly patients with intermediate-II and high-risk disease. The Janus kinase (JAK) inhibitor ruxolitinib is approved in the US and EU for the treatment of patients with intermediate or high-risk MF, including PMF, post-PV-MF, and post-ET-MF. In clinical studies, treatment with ruxolitinib has been shown to reduce spleen volume by International Working Group (IWG) criteria in approximately 28% to 42% of patients and improve constitutional symptoms of MF in approximately 46% of patients (Verstovsek, J Hematol Oncol. 2017). Ruxolitinib provides symptomatic improvement, however, does not target the malignant clone or appreciably reduce the degree of fibrosis; some patients experience disease progression and leukemic transformation while on therapy (Versotvsek, NEJM. 2010; Harrison, NEJM. 2012; Kremyanskaya, Br J Hem. 2014). Moreover, ruxolitinib is associated with AEs including anemia and thrombocytopenia, which can lead to discontinuation. Approximately 50% of patients treated with ruxolitinib discontinued treatment within 3 years and 73% at 5 years (Verstovsek, Haematologica. 2015; Verstovsek, J Hematol Oncol. 2017; Cervantes, Blood. 2013; Harrison, Leukemia. 2016). Median overall survival in patients who discontinue ruxolitinib is 14-16 months, highlighting the need for novel therapies targeting alternative pathways in the setting of failure or intolerance of JAK inhibitor therapy (Newberry, Blood. 2017). The tumor suppressor protein p53 is the master regulator of cell-cycle arrest and apoptosis in response to cellular stress or DNA damage. Murine double minute 2 (MDM2) is a key regulator of p53, inhibiting its activity via ubiquitination, nuclear export, and direct inhibition of transcriptional activity. Increased MDM2 protein expression has been observed in MF CD34+ cells, suggesting that MF might be sensitive to MDM2 inhibition (Lu M, Blood. 2017). KRT-232 is a potent and selective, oral, small molecule drug that targets MDM2 and prevents MDM2-mediated p53 inhibition, allowing p53 to mediate tumor cell-cycle arrest and apoptosis. In MF, TP53 is observed to be wild-type in 96% of MF patients, suggesting MDM2 inhibition could be a successful therapeutic strategy in this disease (Raza, Am J Hematol. 2012). KRT-232 has been investigated as monotherapy and in combination with trametinib or dabrafenib in phase I studies of AML and melanoma; the most common treatment-related adverse events (TRAEs) observed were nausea, diarrhea, vomiting, decreased appetite, anemia, leukopenia, thrombocytopenia, and fatigue. The majority of TRAEs were grade 1 or 2. Methods: KRT-232 is being evaluated in an open-label phase 2 study in patients with MF who relapsed on or are refractory to JAK inhibitors (Figure). Up to 247 patients ≥ 18 years of age, with ECOG performance status ≤ 2, with high-, intermediate-2, or intermediate-1 risk disease by Dynamic International Prognostic System (DIPSS), and failure of prior treatment with JAK inhibitors will be enrolled. The study will be conducted in 2 parts. Part A will identify the recommended dose and schedule by testing varying doses and schedules across 7 treatment cohorts. Part B will evaluate safety and efficacy using the recommended dose and schedule from Part A. The primary endpoint of the study is to determine spleen response at week 24; secondary endpoints include improvement in MPN-SAF Total Symptom Score (weeks 24 and 48), red blood cell (RBC) transfusion independence, and rates of complete remission and partial remission (IWG-ERT and ELN) at week 24. This trial is enrolling at multiple sites in the United States and Europe (NCT03662126, EudraCT: 2018-001671-21). Disclosures Garcia-Delgado: Hospital Virgen De La Victoria Malaga: Employment; Novartis: Consultancy, Speakers Bureau; Celgene: Speakers Bureau. McLornan:Jazz Pharmaceuticals: Honoraria, Speakers Bureau; Novartis: Honoraria. Jourdan:Novartis: Honoraria; Astellas: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Al-Ali:Celgene: Research Funding; Novartis: Consultancy, Honoraria, Research Funding; CTI: Honoraria. Pluta:Freelight Poland: Honoraria; Sandoz: Honoraria; Servier: Honoraria; Jansen-Cilag: Honoraria; Novartis: Honoraria; Takeda: Honoraria; Roche: Honoraria; Specialistic Hospital in Brzozow,Dept of Haematooncology Ks.Bielawskiego 18 36-200 Brzozow, Poland: Employment; Teva: Honoraria; Roche Poland: Membership on an entity's Board of Directors or advisory committees; Jansen Cilag Poland: Membership on an entity's Board of Directors or advisory committees. Ewing:Novartis: Honoraria, Other: Meeting attendance sponsorship ; Bristol Myers-Squibb: Other: Meeting attendance sponsorship . Khan:Amgen: Consultancy; Celgene: Consultancy; Incyte: Honoraria; Pfizer: Consultancy; Takeda: Research Funding. Jost:Novartis: Research Funding; Celgene: Other: Travel Support; Pfizer: Consultancy, Speakers Bureau; Novartis: Consultancy, Speakers Bureau; Abbvie: Consultancy, Patents & Royalties: Royalty payments for the drug compound ABT-199, Research Funding; Bohringer: Consultancy, Research Funding; BMS: Consultancy, Speakers Bureau. Rothbaum:Kartos Therapeutics: Employment, Patents & Royalties: Pending; Quogue Bioventures LLC: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. McGreivy:Kartos Therapeutics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Verstovsek:Incyte: Research Funding; Roche: Research Funding; NS Pharma: Research Funding; Celgene: Consultancy, Research Funding; Gilead: Research Funding; Promedior: Research Funding; CTI BioPharma Corp: Research Funding; Genetech: Research Funding; Blueprint Medicines Corp: Research Funding; Novartis: Consultancy, Research Funding; Sierra Oncology: Research Funding; Pharma Essentia: Research Funding; Astrazeneca: Research Funding; Ital Pharma: Research Funding; Protaganist Therapeutics: Research Funding; Constellation: Consultancy; Pragmatist: Consultancy. OffLabel Disclosure: Yes, KRT-232 is an investigational small molecule MDM2 inhibitor. This trial-in-progress abstract describes a registered clinical trial that will evaluate the safety and efficacy of KRT-232 for patients with myelofibrosis.

Description: The Ly49 natural killer (NK)–cell receptor family comprises both activating and inhibitory members, which recognize major histocompatibility complex (MHC) class I or MHC class I–related molecules and are involved in target recognition. As previously shown, the Ly49E receptor fails to bind to a variety of soluble or cell-bound MHC class I molecules, indicating that its ligand is not an MHC class I molecule. Using BWZ.36 reporter cells, we demonstrate triggering of Ly49E by the completely distinct, non–MHC-related protein urokinase plasminogen activator (uPA). uPA is known to be secreted by a variety of cells, including epithelial and hematopoietic cells, and levels are up-regulated during tissue remodeling, infections, and tumorigenesis. Here we show that addition of uPA to Ly49E-positive adult and fetal NK cells inhibits interferon-γ secretion and reduces their cytotoxic potential, respectively. These uPA-mediated effects are Ly49E-dependent, as they are reversed by addition of anti-Ly49E monoclonal antibody and by down-regulation of Ly49E expression using RNA interference. Our results suggest that uPA, besides its established role in fibrinolysis, tissue remodeling, and tumor metastasis, could be involved in NK cell–mediated immune surveillance and tumor escape.

Description: Natural killer (NK) cell development in the bone marrow is not fully understood. Following lineage commitment, these cells appear to advance through a series of developmental stages that are beginning to be characterized. We previously reported a selective deficiency of NK cells in a C57BL/6 mouse with a transgenic construct consisting of the cDNA for the Ly49A major histocompatibility complex (MHC) class 1–specific inhibitory receptor driven by the granzyme A gene. This mouse has few NK cells in peripheral tissues with relative preservation of other immune cells, including T and B cells. Herein we demonstrate that these mice have an accumulation of NK cells with an immature phenotype in the bone marrow, consistent with a block at a previously proposed stage in normal NK-cell development. The phenotype is associated with transgenic insertion into Atf2, the gene for the basic leucine zipper (bZIP) transcription factor family member ATF-2. Although analysis of Atf2-null NK cells shows no defect, the transgenic mice express abnormal truncated Atf2 transcripts that may mediate a repressor effect because ATF2 can heterodimerize with other bZIP molecules. The defect is cell intrinsic, suggesting that certain bZIP molecules play significant roles in NK-cell development.

Description: Natural killer (NK) cells have important functions in cancer immunosurveillance, BM allograft rejection, fighting infections, tissue homeostasis, and reproduction. NK cell–based therapies are promising treatments for blood cancers. Overcoming their currently limited efficacy requires a better understanding of the molecular mechanisms controlling NK cell development and dampening their effector functions. NK cells recognize the loss of self-antigens or up-regulation of stress-induced ligands on pathogen-infected or tumor cells through invariant NK cell receptors (NKRs), and then kill such stressed cells. Two second-messenger pathways downstream of NKRs are required for NK cell maturation and effector responses: PIP3 generation by PI3K and generation of diacylglycerol and IP3 by phospholipase-Cγ (PLCγ). In the present study, we identify a novel role for the phosphorylated IP3 metabolite inositol (1,3,4,5)tetrakisphosphate (IP4) in NK cells. IP4 promotes NK cell terminal differentiation and acquisition of a mature NKR repertoire. However, in mature NK cells, IP4 limits NKR-induced IFNγ secretion, granule exocytosis, and target-cell killing, in part by inhibiting the PIP3 effector-kinase Akt. This identifies IP4 as an important novel regulator of NK cell development and function and expands our understanding of the therapeutically important mechanisms dampening NK cell responses. Our results further suggest that PI3K regulation by soluble IP4 is a broadly important signaling paradigm.

Description: The differentiation of natural killer (NK) cells and a subpopulation of NK cells which requires an intact thymus, that is, thymic NK cells, is poorly understood. Previous in vitro studies indicate that double negative (CD4−CD8−, DN) thymocytes can develop into cells with NK cell markers, but these cells have not been well characterized. Herein, we generated and characterized NK cells differentiating from thymic DN precursors. Sorted DN1 (CD44+CD25−) CD122−NK1.1− thymocytes from Rag1−/− mice were adoptively transferred into Rag1−/−Ly5.1 congenic mice. After intrathymic injection, donor-derived cells phenotypically resembling thymic NK cells were found. To further study their differentiation, we seeded sorted DN1 CD122−NK1.1− thymocytes on irradiated OP9 bone marrow stromal cells with IL-15, IL-7, Flt3L, and stem cell factor. NK1.1+ cells emerged after 7 days. In vitro differentiated NK cells acquired markers associated with immature bone marrow–derived NK cells, but also expressed CD127, which is typically found on thymic NK cells. Furthermore, we found that in vitro cells generated from thymic precursors secreted cytokines when stimulated and degranulated on target exposure. Together, these data indicate that functional thymic NK cells can develop from a DN1 progenitor cell population.

Description: Natural killer (NK) cells are innate immune cells that mediate resistance against viruses and tumors. They express multiple activating receptors that couple to immunoreceptor tyrosine-based activation motif (ITAM)–containing signaling chains for downstream cell activation. Ligation of activating NK-cell receptors induces NK-cell cytotoxicity and cytokine release. How these distinct events are selectively controlled is not well defined. Here we report the identification of a specific signaling pathway that operates downstream of the ITAM-coupled NK-cell receptors NK1.1, Ly49D, Ly49H, and NKG2D. Using primary NK cells from Bcl10−/−, Malt1−/−, Carma1−/−, and Card9−/− mice, we demonstrate a key role for Bcl10 signalosomes in the activation of canonical NF-κB signaling as well as JNK and p38 MAPK upon NK-cell triggering. Bcl10 directly cooperates with Malt1 and depends on Carma1 (Card11) but not on Card9 for NK-cell activation. These Bcl10-dependent cascades selectively control cytokine and chemokine production but do not affect NK-cell differentiation or killing. Thus, we identify a molecular basis for the segregation of NK-cell receptor–induced signals for cytokine release and target cell killing and extend the previously recognized roles for CARD-protein/Bcl10/Malt1 complexes in ITAM receptor signaling in innate and adaptive immune cells.