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While chimeric antigen receptor (CAR) T cells targeting CD19 can cure a subset of patients with B cell malignancies, most patients treated will not achieve durable remission. Identification of the mechanisms leading to failure is essential to broadening the efficacy of this promising platform. Several studies have demonstrated that disruption of CD19 genes and transcripts can lead to disease relapse after initial response; however, few other tumor-intrinsic drivers of CAR T cell failure have been reported. Here we identify expression of the Golgi-resident intramembrane protease Signal peptide peptidase-like 3 (SPPL3) in malignant B cells as a potent regulator of resistance to CAR therapy. Loss of SPPL3 results in hyperglycosylation of CD19, an alteration that directly inhibits CAR T cell effector function and suppresses anti-tumor cytotoxicity. Alternatively, over-expression of SPPL3 drives loss of CD19 protein, also enabling resistance. In this pre-clinical model these findings identify post-translational modification of CD19 as a mechanism of antigen escape from CAR T cell therapy. Loss of surface CD19 expression by leukemic cells leads to resistance and relapse to CD19-targeted CAR-T therapies. Here the authors show that loss of SPPL3 in malignant B cells results in hyperglycosylation of CD19.

Background The aim of this study is to determine whether Hypoxanthine Guanine Phosphoribosyltransferase (HPRT) could be used as a biomarker for the diagnosis and treatment of B cell malignancies. With 4.3% of all new cancers diagnosed as Non-Hodgkin lymphoma, finding new biomarkers for the treatment of B cell cancers is an ongoing pursuit. HPRT is a nucleotide salvage pathway enzyme responsible for the synthesis of guanine and inosine throughout the cell cycle. Methods Raji cells were used for this analysis due to their high HPRT internal expression. Internal expression was evaluated utilizing western blotting and RNA sequencing. Surface localization was analyzed using flow cytometry, confocal microscopy, and membrane biotinylation. To determine the source of HPRT surface expression, a CRISPR knockdown of HPRT was generated and confirmed using western blotting. To determine clinical significance, patient blood samples were collected and analyzed for HPRT surface localization. Results We found surface localization of HPRT on both Raji cancer cells and in 77% of the malignant ALL samples analyzed and observed no significant expression in healthy cells. Surface expression was confirmed in Raji cells with confocal microscopy, where a direct overlap between HPRT specific antibodies and a membrane-specific dye was observed. HPRT was also detected in biotinylated membranes of Raji cells. Upon HPRT knockdown in Raji cells, we found a significant reduction in surface expression, which shows that the HPRT found on the surface originates from the cells themselves. Finally, we found that cells that had elevated levels of HPRT had a direct correlation to XRCC2, BRCA1, PIK3CA, MSH2, MSH6, WDYHV1, AK7, and BLMH expression and an inverse correlation to PRKD2, PTGS2, TCF7L2, CDH1, IL6R, MC1R, AMPD1, TLR6, and BAK1 expression. Of the 17 genes with significant correlation, 9 are involved in cellular proliferation and DNA synthesis, regulation, and repair. Conclusions As a surface biomarker that is found on malignant cells and not on healthy cells, HPRT could be used as a surface antigen for targeted immunotherapy. In addition, the gene correlations show that HPRT may have an additional role in regulation of cancer proliferation that has not been previously discovered.

The family Anthocoridae (Hemiptera:Heteroptera) contains between 400 and 600 species distributed worldwide, chiefly on the continents but also on oceanic islands. They are small (1.4-4.5 mm) and common to a wide variety of habitats. Many are found in cryptic habitats such as galls, several widespread genera are surface feeders on small arthropods ( Anthocoris, Orius , and Tetraphleps ), and others can be found in ant nests and, especially, under bark. Wing polymorphism is common in this family, often associated with the cryptic habit. Most known species are predaceous, though some take plant food as well (e.g. Orius insidiosus, Orius pallidicornis ). A few of these are believed to be entirely phytophagous ( Paratriphleps laeviusculus ). Their small size and often generalized feeding habits have resulted in about 30 introduced species, mostly accidental. A few have been introduced deliberately as biological control agents ( Anthocoris spp., Montandoniola moraguesi, O. insidiosus, Orius tristicolor , and Tetraphleps spp.). Most nonindigenous species seem to have been distributed as a result of human activities, especially commerce. The predaceous habits of many Anthocoridae have attracted the attention of researchers who work in agroecosystems. Integrated pest management programs often include these predators, which has given us greater knowledge about these species than those found in natural ecosystems. Exciting discoveries about the attractiveness to these bugs of certain volatile plant and arthropod compounds are opening new areas of investigation into their chemical ecology. The reactions of these tiny predators will surely become better understood as a result.

BackgroundCAR T cell therapy is a promising therapeutic modality for cancer treatment, but poor CAR T cell persistence limits efficacy in patients.1 CAR T cells with a memory-like phenotype are associated with durable persistence in patients and response to therapy,2 thereby implicating memory as an important therapeutic axis. Thus, strategies to stably promote CAR T memory differentiation are urgently needed. Here, we demonstrate that FOXO1 is required for the development of memory CAR T cells and that FOXO1 overexpression maintains memory gene expression programs and enhances CAR T cell antitumor activity in liquid and solid tumor models.MethodsT cells were co-transduced to express a CAR and a transcription factor, and in relevant experiments were CRISPR-edited using Cas9. CAR T cell function was assessed in vitro using tumor co-culture assays wherein cytokine secretion, killing, and metabolic fitness (via Seahorse) were measured. In vivo studies were performed using murine xenograft tumor models in NSG mice. CAR T phenotyping experiments were performed using flow cytometry, RNA-seq, and ATAC-seq. Public datasets were reanalyzed to evaluate FOXO1 activity in patients treated with CAR T cell therapy.ResultsFOXO1 knockout in human CAR T cells prevented the development of a memory-like phenotype, and instead, promoted an exhausted phenotype and gene expression profile and attenuated antitumor activity in vitro and in vivo. FOXO1 overexpression in CAR T cells dramatically enhanced functionality in vitro and in vivo, especially in models of chronic antigen stimulation, and promoted a memory-like phenotype via flow cytometry, RNA-seq, and ATAC-seq. A FOXO1 regulon consisting of 41 putative FOXO1 target genes, which were unbiasedly identified in our knockout and overexpression studies, highly correlated with long-term persistence and positive outcomes in patients treated with CD19 CAR T cells.ConclusionsOur results demonstrate that FOXO1 is a master regulator of CAR T cell memory programming and that overexpression of FOXO1 triggers both transcriptional and epigenetic changes in CAR T cells that enhance memory differentiation, persistence, and potency. Our findings further demonstrate the potential for transcription factor engineering as an approach to generate highly effective CAR T cell products for antitumor therapy.ReferencesWeber EW, Maus MV, Mackall CL. The Emerging Landscape of Immune Cell Therapies. Cell. 2020;181:46–62.Fraietta JA, et al. Determinants of response and resistance to CD19 chimeric antigen receptor (CAR) T cell therapy of chronic lymphocytic leukemia. Nat. Med. 2018;24:563–571.Ethics ApprovalAll animal studies were undertaken under Stanford University APLAC #31287 and Children’s Hospital of Philadelphia ACUP-approved protocols.

Chimeric antigen receptor (CAR) T cells mediate anti-tumour effects in a small subset of patients with cancer 1 – 3 , but dysfunction due to T cell exhaustion is an important barrier to progress 4 – 6 . To investigate the biology of exhaustion in human T cells expressing CAR receptors, we used a model system with a tonically signaling CAR, which induces hallmark features of exhaustion 6 . Exhaustion was associated with a profound defect in the production of IL-2, along with increased chromatin accessibility of AP-1 transcription factor motifs and overexpression of the bZIP and IRF transcription factors that have been implicated in mediating dysfunction in exhausted T cells 7 – 10 . Here we show that CAR T cells engineered to overexpress the canonical AP-1 factor c-Jun have enhanced expansion potential, increased functional capacity, diminished terminal differentiation and improved anti-tumour potency in five different mouse tumour models in vivo. We conclude that a functional deficiency in c-Jun mediates dysfunction in exhausted human T cells, and that engineering CAR T cells to overexpress c-Jun renders them resistant to exhaustion, thereby addressing a major barrier to progress for this emerging class of therapeutic agents. Chimeric antigen receptor (CAR) T cells engineered to overexpress the canonical AP-1 transcription factor c-Jun are resistant to T cell exhaustion, and provide enhanced therapeutic benefit in mouse tumour models.

The disialoganglioside GD2 is overexpressed on several solid tumors, and monoclonal antibodies targeting GD2 have substantially improved outcomes for children with high-risk neuroblastoma. However, approximately 40% of patients with neuroblastoma still relapse, and anti-GD2 has not mediated significant clinical activity in any other GD2 + malignancy. Macrophages are important mediators of anti-tumor immunity, but tumors resist macrophage phagocytosis through expression of the checkpoint molecule CD47, a so-called ‘Don’t eat me’ signal. In this study, we establish potent synergy for the combination of anti-GD2 and anti-CD47 in syngeneic and xenograft mouse models of neuroblastoma, where the combination eradicates tumors, as well as osteosarcoma and small-cell lung cancer, where the combination significantly reduces tumor burden and extends survival. This synergy is driven by two GD2-specific factors that reorient the balance of macrophage activity. Ligation of GD2 on tumor cells (a) causes upregulation of surface calreticulin, a pro-phagocytic ‘Eat me’ signal that primes cells for removal and (b) interrupts the interaction of GD2 with its newly identified ligand, the inhibitory immunoreceptor Siglec-7. This work credentials the combination of anti-GD2 and anti-CD47 for clinical translation and suggests that CD47 blockade will be most efficacious in combination with monoclonal antibodies that alter additional pro- and anti-phagocytic signals within the tumor microenvironment. The combination of anti-GD2 and CD47 blockade mediates robust anti-tumor activity in mouse models of neuroblastoma, osteosarcoma and small-cell lung cancer by reorienting macrophage activity toward tumor cell phagocytosis.

A major limitation of chimeric antigen receptor (CAR) T cell therapies is the poor persistence of these cells in vivo 1 . The expression of memory-associated genes in CAR T cells is linked to their long-term persistence in patients and clinical efficacy 2 – 6 , suggesting that memory programs may underpin durable CAR T cell function. Here we show that the transcription factor FOXO1 is responsible for promoting memory and restraining exhaustion in human CAR T cells. Pharmacological inhibition or gene editing of endogenous FOXO1 diminished the expression of memory-associated genes, promoted an exhaustion-like phenotype and impaired the antitumour activity of CAR T cells. Overexpression of FOXO1 induced a gene-expression program consistent with T cell memory and increased chromatin accessibility at FOXO1-binding motifs. CAR T cells that overexpressed FOXO1 retained their function, memory potential and metabolic fitness in settings of chronic stimulation, and exhibited enhanced persistence and tumour control in vivo. By contrast, overexpression of TCF1 (encoded by TCF7 ) did not enforce canonical memory programs or enhance the potency of CAR T cells. Notably, FOXO1 activity correlated with positive clinical outcomes of patients treated with CAR T cells or tumour-infiltrating lymphocytes, underscoring the clinical relevance of FOXO1 in cancer immunotherapy. Our results show that overexpressing FOXO1 can increase the antitumour activity of human CAR T cells, and highlight memory reprogramming as a broadly applicable approach for optimizing therapeutic T cell states. The transcription factor FOXO1 has a key role in human T cell memory, and manipulating FOXO1 expression could provide a way to enhance CAR T cell therapies by increasing CAR T cell persistence and antitumour activity.

Background Belize is a middle-income Caribbean country with poorly described cancer epidemiology and no comprehensive cancer care capacity. In 2018, GO, Inc., a US-based NGO, partnered with the Ministry of Health and the national hospital in Belize City to create the first public oncology clinic in the country. Here, we report demographics from the clinic and describe time intervals to care milestones to allow for public health targeting of gaps. Patients and Methods Using paper charts and a mobile health platform, we performed a retrospective chart review at the Karl Heusner Memorial Hospital (KHMH) clinic from 2018 to 2022. Results During this time period, 465 patients with cancer presented to the clinic. Breast cancer (28%) and cervical cancer (12%) were most common. Most patients (68%) presented with stage 3 or 4 disease and were uninsured (78%) and unemployed (79%). Only 21% of patients ever started curative intent treatment. Median time from patient-reported symptoms to a biopsy or treatment was 130 and 189 days. For the most common cancer, breast, similar times were seen at 140 and 178 days. Time intervals at the clinic: <30 days from initial visit to biopsy (if not previously performed) and <30 days to starting chemotherapy. Conclusion This study reports the first clinic-based cancer statistics for Belize. Many patients have months between symptom onset and treatment. In this setting, the clinic has built infrastructure allowing for minimal delays in care despite an underserved population. This further affirms the need for infrastructure investment and early detection programs to improve outcomes in Belize. In 2018, the first public oncology clinic was established in Belize. This article reports demographics from the clinic and describes time intervals to care milestones to allow for public health targeting of gaps and provide a roadmap for public cancer care in the country.

Old-growth forests of the Pacific Northwest extend along the coastal region from southern Alaska to northern California and are composed largely of conifer rather than hardwood tree species. Many of these trees achieve great age (500-1,000 yr). Natural succession that follows forest stand destruction normally takes over 100 years to reach the young mature forest stage. This succession may continue on into old-growth for centuries. The changing structural complexity of the forest over time, combined with the many different plant species that characterize succession, results in an array of arthropod habitats. It is estimated that 6,000 arthropod species may be found in such forests-over 3,400 different species are known from a single 6,400 ha site in Oregon. Our knowledge of these species is still rudimentary and much additional work is needed throughout this vast region. Many of these species play critical roles in the dynamics of forest ecosystems. They are important in nutrient cycling, as herbivores, as natural predators and parasites of other arthropod species. This faunal diversity reflects the diversity of the environment and the arthropod complex provides a sensitive barometer of the conditions of the forest. Conservation efforts for forest arthropods are limited at present and controlled largely by land-use policies. For example, an effort is being made to include arthropods in conservation efforts for the Northern Spotted Owl and arthropods will be included in the Forest Health Monitoring program now underway by the U.S. Forest Service. Evidence from other parts of the world suggest that arthropods that depend upon large pieces of dead wood may be particularly threatened by forest management practices. Much remains to be done in the conservation of forest arthropods.