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Lisocabtagene maraleucel (liso-cel) is an investigational drug product composed of autologous CD8+ and CD4+ T cells expressing a CD19-specific chimeric antigen receptor (CAR), being evaluated in clinical trials for treatment of B cell malignancies. The CELMoD CC-122 is being evaluated in non-Hodgkin lymphoma (NHL) and can augment T cell function through Cereblon-mediated degradation of Ikaros and Aiolos. Combination approaches aimed at enhancing CAR T cell function may increase the rate, depth, and durability of clinical responses. Here we report pre-clinical results supporting the potential combination of CC-122 an liso-cel. To determine potentiating effects of CC-122 following acute activation (anti-idiotypic Ab or CD19+ targets), T cells expressing the CAR of liso-cel were cultured with CC-122 and assessed for intracellular signaling, cytolytic activity, cytokine production, and activation marker expression. CC-122 significantly increased NFκB, Nur77, and STAT5 as well as effector cytokine production and activation marker expression. Activation-induced exhaustion of CAR T may decrease durable responses in patients. We developed a chronic stimulation assay to render CAR T cells hypofunctional (reduced cytolysis and IL-2 secretion) and examined whether enhanced activation mediated by CC-122 would exhaust the CD19-directed CAR T cells more rapidly. CC-122 was added in one of two regimens: during chronic stimulation (“concurrent”) or during re-challenge (“rescue”). Effects on exhaustion in “concurrent” or “rescue” cultures were evaluated, including by RNAseq and measuring of effector function in re-challenge assays with CD19+ targets. Surprisingly, “concurrent” CC-122 partially reversed a gene signature associated with CAR T cell hypofunctionality and preserved more effector function relative to controls. In “rescue” assays, CC-122 dose-dependently increased cytokine production and restored cytolytic function of exhausted CAR T cells upon re-challenge with CD19+ tumor spheroids. In a disseminated tumor xenograft model, concurrent or delayed dosing of CC-122, in combination with two sub-curative doses of liso-cel, significantly increased median survival by 30 to 40 days. In some cases, survival endpoints were not reached with the combination. Taken together, CC-122 treatment of CD19-directed CAR T cells was shown to enhance CAR T activation in acute stimulation assays; limit CAR T exhaustion onset during chronic stimulation as assessed by gene expression; and reverse an exhausted, hypofunctional CAR T state induced by chronic stimulation. The data suggest that liso-cel pharmacologic activity may both be enhanced and extended by this combinatorial approach. These findings support an ongoing clinical investigation evaluating the addition of CC-122 to liso-cel in patients with R/R, aggressive NHL (NCT03310619). Citation Format: Heidi K. Jessup, Evan P. Thomas, Jim S. Qin, Yue Jiang, Oleksandr Baturevych, Neha Soni, Aye T. Chen, Ruth A. Salmon, Melissa G. Works, Michael O. Ports. Avadomide (CC-122) increases effector function and reverses exhaustion in chronically stimulated lisocabtagene maraleucel anti-CD19 CAR T drug product [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2320.

In a forward genetic approach to identify novel genes for congenital muscle diseases, a zebrafish mutant, designated patchytail, was identified that exhibits degenerating muscle fibers with impaired motility behavior. Genetic mapping identified a genomic locus containing the zebrafish ortholog of the dystroglycan gene (DAG1). Patchytail fish contain a point mutation (c.1700T>A) in dag1, resulting in a missense change p.V567D. This change is associated with reduced transcripts and a complete absence of protein. The absence of α-dystroglycan and β-dystroglycan caused destabilization of dystroglycan complex, resulting in membrane damages. Membrane damage was localized on the extracellular matrix at myosepta as well as basement membrane between adjacent myofibers. These studies also identified structural abnormalities in triads at 3 days post fertilization (dpf) of dystroglycan-deficient muscles, significantly preceding sarcolemmal damage that becomes evident at 7 dpf. Immunofluorescence studies identified a subpopulation of dystroglycan that is expressed at t-tubules in normal skeletal muscles. In dag1-mutated fish, smaller and irregular-shaped t-tubule vesicles, as well as highly disorganized terminal cisternae of sarcoplasmic reticulum, were common. In addition to skeletal muscle defects, dag1-mutated fish have brain abnormalities and ocular defects in posterior as well as anterior chambers. These phenotypes of dystroglycan-deficient fish are highly reminiscent of the phenotypes observed in the human conditions muscle–eye–brain disease and Walker–Warburg syndrome. This animal model will provide unique opportunities in the understanding of biological functions of dystroglycan in a wide range of dystroglycanopathies, as disruption of this gene in higher vertebrates results in early embryonic lethality.

Congenital muscular dystrophy (CMD) is a clinically and genetically heterogeneous group of inherited muscle disorders. In patients, muscle weakness is usually present at or shortly after birth and is progressive in nature. Merosin deficient congenital muscular dystrophy (MDC1A) is a form of CMD caused by a defect in the laminin-α2 gene (LAMA2). Laminin-α2 is an extracellular matrix protein that interacts with the dystrophin-dystroglycan (DGC) complex in membranes providing stability to muscle fibers. In an N-ethyl-N-nitrosourea mutagenesis screen to develop zebrafish models of neuromuscular diseases, we identified a mutant fish that exhibits severe muscular dystrophy early in development. Genetic mapping identified a splice site mutation in the lama2 gene. This splice site is highly conserved in humans and this mutation results in mis-splicing of RNA and a loss of protein function. Homozygous lama2 mutant zebrafish, designated lama2(cl501/cl501), exhibited reduced motor function and progressive degeneration of skeletal muscles and died at 8-15 days post fertilization. The skeletal muscles exhibited damaged myosepta and detachment of myofibers in the affected fish. Laminin-α2 deficiency also resulted in growth defects in the brain and eye of the mutant fish. This laminin-α2 deficient mutant fish represents a novel disease model to develop therapies for modulating splicing defects in congenital muscular dystrophies and to restore the muscle function in human patients with CMD.

Evaluating hematopoietic stem cell (HSC) function in vivo requires a long-term transplantation assay. Although zebrafish are a powerful model for discovering the genetics of hematopoiesis, hematopoietic transplantation approaches have been underdeveloped. Here we established a long-term reconstitution assay in adult zebrafish. Primary and secondary recipients showed multilineage engraftment at 3 months after transplantation. Limiting dilution data suggest that at least 1 in 65 000 zebrafish marrow cells contain repopulating activity, consistent with mammalian HSC frequencies. We defined zebrafish haplotypes at the proposed major histocompatibility complex locus on chromosome 19 and tested functional significance through hematopoietic transplantation. Matching donors and recipients dramatically increased engraftment and percentage donor chimerism compared with unmatched fish. These data constitute the first functional test of zebrafish histocompatibility genes, enabling the development of matched hematopoietic transplantations. This lays the foundation for competitive transplantation experiments with mutant zebrafish HSCs and chemicals to test for effects on engraftment, thereby providing a model for human hematopoietic diseases and treatments not previously available.

A comprehensive understanding of the genes and pathways regulating hematopoiesis is needed to identify genes causally related to bone marrow failure syndromes, myelodysplastic syndromes, and hematopoietic neoplasms. To identify novel genes involved in hematopoiesis, we performed an ethyl-nitrosourea mutagenesis screen in zebrafish (Danio rerio) to search for mutants with defective definitive hematopoiesis. We report the recovery and analysis of the grechetto mutant, which harbors an inactivating mutation in cleavage and polyadenylation specificity factor 1 (cpsf1), a gene ubiquitously expressed and required for 3′ untranslated region processing of a subset of pre-mRNAs. grechetto mutants undergo normal primitive hematopoiesis and specify appropriate numbers of definitive HSCs at 36 hours postfertilization. However, when HSCs migrate to the caudal hematopoietic tissue at 3 days postfertilization, their numbers start decreasing as a result of apoptotic cell death. Consistent with Cpsf1 function, c-myb:EGFP+ cells in grechetto mutants also show defective polyadenylation of snrnp70, a gene required for HSC development. By 5 days postfertilization, definitive hematopoiesis is compromised and severely decreased blood cell numbers are observed across the myeloid, erythroid, and lymphoid cell lineages. These studies show that cpsf1 is essential for HSC survival and differentiation in caudal hematopoietic tissue.

Within the past two decades, the zebrafish (Danio rerio) has become an excellent model to study the development of hematopoietic stem cells (HSCs). All vertebrates including zebrafish have primitive and definitive waves of hematopoiesis, but self-renewing pluripotent HSCs are only produced by the definitive wave. The primitive wave occurs in two intraembryonic locations called the intermediate cell mass (ICM) and the anterior lateral mesoderm (ALM). Primitive erythropoiesis is in the ICM, whereas myelopoiesis initiates in the ALM. After circulation starts at 24 h post-fertilization, hematopoiesis shifts to the posterior blood island (PBI) for a brief period. The definitive wave starts in the aorta-gonad-mesonephros (AGM). There are three different HSC migration and colonization events that begin 2 days post-fertilization: AGM progenitor cells migrate to (1) the caudal hematopoietic tissue (CHT), which is an intermediate site of blood development; (2) the thymus, which is a site of lymphocyte maturation; and (3) the developing kidney marrow, which is the larval and adult location for production of all hematopoietic cell types, and is comparable to the bone marrow of mammals. Many of the transcription factors and signaling pathways that regulate the formation of HSCs in a zebrafish are conserved with mammals. Large-scale forward and reverse genetic screens have identified zebrafish blood and HSC mutants that represent models for known human diseases. Along with the technological advancements in the field of zebrafish research, future HSC studies in zebrafish will help us illuminate the genetic network controlling the development and function of stem cells in all vertebrates.

Abstract 1606 Hematopoiesis is a tightly regulated process requiring the differential activation of specific genetic programs. Inactivating mutations of genes involved in normal hematopoiesis have been implicated in bone marrow failure syndromes, myelodysplastic syndromes and hematopoietic neoplasms. Despite recent advances, many genes regulating normal hematopoiesis are still unknown and innovative gene-discovery approaches can benefit our understanding of the molecular mechanisms underlying this complex developmental process. The combined genetic and embryologic strengths of the zebrafish model system are ideal for studying factors regulating hematopoiesis, which is highly conserved with mammals. We have used ENU-mutated zebrafish lines to screen for genes whose inactivation results in the loss of definitive hematopoiesis. Here, we report the identification and analysis of the zebrafish mutant grechetto, caused by a lethal, recessive, inactivating mutation (grcl8a12) of the cleavage and specificity factor 1 (cpsf1) gene. Cpsf1 encodes a protein required for processing the 3’UTR of a subset of pre-mRNAs. Cpsf1 is maternally expressed ubiquitously through the first five days of development including primitive myeloid and erythroid cells and definitive hematopoietic stem cells (HSC). Although grechetto mutants fail to express cspf1 already at 24 hpf, they appear to develop normally until 72 hpf, when they first become morphologically distinct from wild-type siblings with a smaller head and the absence of a protruding jaw. Importantly, primitive hematopoiesis appears normal, as shown by expression of the myeloid and erythroid markers mpx and band3, respectively, at 24 and 48 hpf by whole mount RNA in situ hybridization (WISH). By 120 hpf the phenotype is more marked and embryos have a curved body, cardiac edema, development defects in the jaw and gut and a reduction in the number of iridophores. While many of these affected tissues represent derivatives of the neural crest (NC), the expression of markers of NC specification (sox10 and foxd3 at 15,5 hpf, crestin at 24 hpf), migration (dlx2 at 36 and 48 hpf) and differentiation (pax9a at 48 hpf) are normal. When definitive hematopoiesis was assayed for markers of mature blood cells by WISH at 120 hpf, grechetto mutants exhibit a loss of myeloid (mpx, lysC, l-plastin), erythroid (band3, gata1) and lymphoid cells (rag1, lck). Since myeloid and erythroid cells are normal at 24 hpf, but severely reduced at 120 hpf, we investigated whether the absence of HSC could explain the loss of definitive hematopoiesis in grechetto mutants. Interestingly, WISH staining for the HSC marker c-myb showed that in mutant embryos HSCs are normally specified and represented in the ventral wall of the dorsal aorta at 36 and 48 hpf. Nevertheless, at 72 hpf the numbers of HSCs are reduced in grechetto mutants upon their migration to the caudal hematopoietic tissue (CHT) and by 120 hpf very few remain. To investigate the fate of these HSCs we crossed the grechetto line with the Tg(c-myb:EGFP) reporter line, that expresses EGFP under the control of the c-myb promoter. By whole mount immunostaining, we were able to find increased co-localization of activated caspase3 and TUNEL staining in the c-myb:EGFP positive cells in the CHT in grechetto mutants compared to WT siblings at 72 and 96 hpf, suggesting that HSCs undergo apoptosis during this stage. Furthermore, PI cell cycle profile of sorted c-myb:EGFP+ cells from 96 hpf mutants shows a sub-G1 peak, representing pyknotic cell nuclei undergoing advanced stages of apoptosis. Importantly, both activated caspase3 staining and TUNEL assay did not show increased apoptosis in the tissue immediately surrounding the CHT. These studies show that cpsf1 is not required for primitive hematopoiesis or definitive HSC specification, however, it is essential for HSC survival in the CHT. The fact that grechetto embryos do not undergo generalized apoptotic cell death suggests that, despite ubiquitous cpsf1 expression and the general development defects of grechetto mutants, the prosurvival pathways in the HSC compartment are particularly dependent on Cpsf1 activity. Disclosures: Zon: FATE, Inc.: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Stemgent: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.