NOTCH1 signaling during CD4+ T-cell activation alters transcription factor networks and enhances antigen responsiveness.

Adoptive transfer of T cells expressing chimeric antigen receptors (CAR-T) effectively treats refractory hematologic malignancies in a subset of patients but can be limited by poor T-cell expansion and persistence in vivo. Less differentiated T-cell states correlate with the capacity of CAR-T to proliferate and mediate antitumor responses, and interventions that limit tumor-specific T-cell differentiation during ex vivo manufacturing enhance efficacy. NOTCH signaling is involved in fate decisions across diverse cell lineages and in memory CD8+ T cells was reported to upregulate the transcription factor FOXM1, attenuate differentiation, and enhance proliferation and antitumor efficacy in vivo. Here, we used a cell-free culture system to provide an agonistic NOTCH1 signal during naïve CD4+ T-cell activation and CAR-T production and studied the effects on differentiation, transcription factor expression, cytokine production, and responses to tumor. NOTCH1 agonism efficiently induced a stem cell memory phenotype in CAR-T derived from naïve but not memory CD4+ T cells and upregulated expression of AhR and c-MAF, driving heightened production of interleukin-22, interleukin-10, and granzyme B. NOTCH1-agonized CD4+ CAR-T demonstrated enhanced antigen responsiveness and proliferated to strikingly higher frequencies in mice bearing human lymphoma xenografts. NOTCH1-agonized CD4+ CAR-T also provided superior help to cotransferred CD8+ CAR-T, driving improved expansion and curative antitumor responses in vivo at low CAR-T doses. Our data expand the mechanisms by which NOTCH can shape CD4+ T-cell behavior and demonstrate that activating NOTCH1 signaling during genetic modification ex vivo is a potential strategy for enhancing the function of T cells engineered with tumor-targeting receptors.

Activating PAX gene family paralogs to complement PAX5 leukemia driver mutations.

PAX5, one of nine members of the mammalian paired box (PAX) family of transcription factors, plays an important role in B cell development. Approximately one-third of individuals with pre-B acute lymphoblastic leukemia (ALL) acquire heterozygous inactivating mutations of PAX5 in malignant cells, and heterozygous germline loss-of-function PAX5 mutations cause autosomal dominant predisposition to ALL. At least in mice, Pax5 is required for pre-B cell maturation, and leukemic remission occurs when Pax5 expression is restored in a Pax5-deficient mouse model of ALL. Together, these observations indicate that PAX5 deficiency reversibly drives leukemogenesis. PAX5 and its two most closely related paralogs, PAX2 and PAX8, which are not mutated in ALL, exhibit overlapping expression and function redundantly during embryonic development. However, PAX5 alone is expressed in lymphocytes, while PAX2 and PAX8 are predominantly specific to kidney and thyroid, respectively. We show that forced expression of PAX2 or PAX8 complements PAX5 loss-of-function mutation in ALL cells as determined by modulation of PAX5 target genes, restoration of immunophenotypic and morphological differentiation, and, ultimately, reduction of replicative potential. Activation of PAX5 paralogs, PAX2 or PAX8, ordinarily silenced in lymphocytes, may therefore represent a novel approach for treating PAX5-deficient ALL. In pursuit of this strategy, we took advantage of the fact that, in kidney, PAX2 is upregulated by extracellular hyperosmolarity. We found that hyperosmolarity, at potentially clinically achievable levels, transcriptionally activates endogenous PAX2 in ALL cells via a mechanism dependent on NFAT5, a transcription factor coordinating response to hyperosmolarity. We also found that hyperosmolarity upregulates residual wild type PAX5 expression in ALL cells and modulates gene expression, including in PAX5-mutant primary ALL cells. These findings specifically demonstrate that osmosensing pathways may represent a new therapeutic target for ALL and more broadly point toward the possibility of using gene paralogs to rescue mutations driving cancer and other diseases.

Inactive ERBB receptors cooperate with reactive oxygen species to suppress cancer progression.

The ERBB receptors are a family of heterodimerization partners capable of driving transformation and metastasis. While the therapeutic targeting of single receptors has proven efficacious, optimal targeting of this receptor family should target all oncogenic members simultaneously. The juxtamembrane domains of ERBB1, ERBB2, and ERBB3 are highly conserved and control various aspects of ERBB-dependent biology. In an effort to block those functions, we have targeted this domain with decoy peptides synthesized in tandem with a cell-penetrating peptide, termed EJ1. Treatment with EJ1 induces cell death, promotes the formation of inactive ERBB multimers, and results in simultaneous reduction of ERBB1, ERBB2, and ERBB3 activation. Treatment also results in the activation of myosin light chain-dependent cell blebbing while inactivating CaMKII signaling, coincident with the induction of cell death. EJ1 also directly translocates to mitochondria, correlating with a loss of mitochondrial membrane potential and production of reactive oxygen species. Finally, treatment of a mouse model of breast cancer with EJ1 results in the inhibition of tumor growth and metastasis without associated toxicities in normal cells. Overall, these data demonstrate that a portion of the ERBB jxm domain, when used as an intracellular decoy, can inhibit tumor growth and metastasis, representing a novel anticancer therapeutic.

Genome-wide atlas of gene expression in the adult mouse brain.

Molecular approaches to understanding the functional circuitry of the nervous system promise new insights into the relationship between genes, brain and behaviour. The cellular diversity of the brain necessitates a cellular resolution approach towards understanding the functional genomics of the nervous system. We describe here an anatomically comprehensive digital atlas containing the expression patterns of approximately 20,000 genes in the adult mouse brain. Data were generated using automated high-throughput procedures for in situ hybridization and data acquisition, and are publicly accessible online. Newly developed image-based informatics tools allow global genome-scale structural analysis and cross-correlation, as well as identification of regionally enriched genes. Unbiased fine-resolution analysis has identified highly specific cellular markers as well as extensive evidence of cellular heterogeneity not evident in classical neuroanatomical atlases. This highly standardized atlas provides an open, primary data resource for a wide variety of further studies concerning brain organization and function.

Restoring RUNX1 deficiency in RUNX1 familial platelet disorder by inhibiting its degradation.

RUNX1 familial platelet disorder (RUNX1-FPD) is an autosomal dominant disorder caused by a monoallelic mutation of RUNX1, initially resulting in approximately half-normal RUNX1 activity. Clinical features include thrombocytopenia, platelet functional defects, and a predisposition to leukemia. RUNX1 is rapidly degraded through the ubiquitin-proteasome pathway. Moreover, it may autoregulate its expression. A predicted kinetic property of autoregulatory circuits is that transient perturbations of steady-state levels result in continued maintenance of expression at adjusted levels, even after inhibitors of degradation or inducers of transcription are withdrawn, suggesting that transient inhibition of RUNX1 degradation may have prolonged effects. We hypothesized that pharmacological inhibition of RUNX1 protein degradation could normalize RUNX1 protein levels, restore the number of platelets and their function, and potentially delay or prevent malignant transformation. In this study, we evaluated cell lines, induced pluripotent stem cells derived from patients with RUNX1-FPD, RUNX1-FPD primary bone marrow cells, and acute myeloid leukemia blood cells from patients with RUNX1 mutations. The results showed that, in some circumstances, transient expression of exogenous RUNX1 or inhibition of steps leading to RUNX1 ubiquitylation and proteasomal degradation restored RUNX1 levels, thereby advancing megakaryocytic differentiation in vitro. Thus, drugs retarding RUNX1 proteolytic degradation may represent a therapeutic avenue for treating bleeding complications and preventing leukemia in RUNX1-FPD.

The copper chelator ATN-224 induces caspase-independent cell death in diffuse large B cell lymphoma.

Bcl-2 and other anti-apoptotic proteins are associated with defective caspase-dependent apoptotic pathways, resulting in chemoresistance. We have previously shown that ATN-224, a copper chelator drug, induces cell death in murine thymic lymphoma cells transfected with Bcl-2. In the current study, we tested whether ATN-224 was effective in diffuse large B cell lymphoma (DLBCL) cells, which have increased anti­apoptotic proteins through translocation or amplification. We found that nanomolar concentrations of ATN-224 induced cell death in DLBCL cells independent of Bcl-2, Bcl-xL or Mcl-1 status. ATN-224 treatment resulted in mitochondrial dysfunction, release of apoptosis-inducing factor (AIF) and induction of caspase-independent cell death. In addition, ATN-224 degraded Mcl-1 and enhanced the effect of the BH3 mimetic ABT-263. These findings indicate that ATN-224 has potential as a therapeutic for the treatment of DLBCL. Induction of caspase­independent cell death in apoptosis­resistant DLBCL would provide a therapeutic alternative for the treatment of refractory disease.