Modular pooled discovery of synthetic knockin sequences to program durable cell therapies.

Chronic stimulation can cause T cell dysfunction and limit the efficacy of cellular immunotherapies. Improved methods are required to compare large numbers of synthetic knockin (KI) sequences to reprogram cell functions. Here, we developed modular pooled KI screening (ModPoKI), an adaptable platform for modular construction of DNA KI libraries using barcoded multicistronic adaptors. We built two ModPoKI libraries of 100 transcription factors (TFs) and 129 natural and synthetic surface receptors (SRs). Over 30 ModPoKI screens across human TCR- and CAR-T cells in diverse conditions identified a transcription factor AP4 (TFAP4) construct that enhanced fitness of chronically stimulated CAR-T cells and anti-cancer function in vitro and in vivo. ModPoKI's modularity allowed us to generate an ∼10,000-member library of TF combinations. Non-viral KI of a combined BATF-TFAP4 polycistronic construct enhanced fitness. Overexpressed BATF and TFAP4 co-occupy and regulate key gene targets to reprogram T cell function. ModPoKI facilitates the discovery of complex gene constructs to program cellular functions.

Genome-wide CRISPR Screens in Primary Human T Cells Reveal Key Regulators of Immune Function.

Human T cells are central effectors of immunity and cancer immunotherapy. CRISPR-based functional studies in T cells could prioritize novel targets for drug development and improve the design of genetically reprogrammed cell-based therapies. However, large-scale CRISPR screens have been challenging in primary human cells. We developed a new method, single guide RNA (sgRNA) lentiviral infection with Cas9 protein electroporation (SLICE), to identify regulators of stimulation responses in primary human T cells. Genome-wide loss-of-function screens identified essential T cell receptor signaling components and genes that negatively tune proliferation following stimulation. Targeted ablation of individual candidate genes characterized hits and identified perturbations that enhanced cancer cell killing. SLICE coupled with single-cell RNA sequencing (RNA-seq) revealed signature stimulation-response gene programs altered by key genetic perturbations. SLICE genome-wide screening was also adaptable to identify mediators of immunosuppression, revealing genes controlling responses to adenosine signaling. The SLICE platform enables unbiased discovery and characterization of functional gene targets in primary cells.

RASA2 ablation in T cells boosts antigen sensitivity and long-term function.

The efficacy of adoptive T cell therapies for cancer treatment can be limited by suppressive signals from both extrinsic factors and intrinsic inhibitory checkpoints1,2. Targeted gene editing has the potential to overcome these limitations and enhance T cell therapeutic function3-10. Here we performed multiple genome-wide CRISPR knock-out screens under different immunosuppressive conditions to identify genes that can be targeted to prevent T cell dysfunction. These screens converged on RASA2, a RAS GTPase-activating protein (RasGAP) that we identify as a signalling checkpoint in human T cells, which is downregulated upon acute T cell receptor stimulation and can increase gradually with chronic antigen exposure. RASA2 ablation enhanced MAPK signalling and chimeric antigen receptor (CAR) T cell cytolytic activity in response to target antigen. Repeated tumour antigen stimulations in vitro revealed that RASA2-deficient T cells show increased activation, cytokine production and metabolic activity compared with control cells, and show a marked advantage in persistent cancer cell killing. RASA2-knockout CAR T cells had a competitive fitness advantage over control cells in the bone marrow in a mouse model of leukaemia. Ablation of RASA2 in multiple preclinical models of T cell receptor and CAR T cell therapies prolonged survival in mice xenografted with either liquid or solid tumours. Together, our findings highlight RASA2 as a promising target to enhance both persistence and effector function in T cell therapies for cancer treatment.

Reprogramming human T cell function and specificity with non-viral genome targeting.

Decades of work have aimed to genetically reprogram T cells for therapeutic purposes1,2 using recombinant viral vectors, which do not target transgenes to specific genomic sites3,4. The need for viral vectors has slowed down research and clinical use as their manufacturing and testing is lengthy and expensive. Genome editing brought the promise of specific and efficient insertion of large transgenes into target cells using homology-directed repair5,6. Here we developed a CRISPR-Cas9 genome-targeting system that does not require viral vectors, allowing rapid and efficient insertion of large DNA sequences (greater than one kilobase) at specific sites in the genomes of primary human T cells, while preserving cell viability and function. This permits individual or multiplexed modification of endogenous genes. First, we applied this strategy to correct a pathogenic IL2RA mutation in cells from patients with monogenic autoimmune disease, and demonstrate improved signalling function. Second, we replaced the endogenous T cell receptor (TCR) locus with a new TCR that redirected T cells to a cancer antigen. The resulting TCR-engineered T cells specifically recognized tumour antigens and mounted productive anti-tumour cell responses in vitro and in vivo. Together, these studies provide preclinical evidence that non-viral genome targeting can enable rapid and flexible experimental manipulation and therapeutic engineering of primary human immune cells.

Hypoxia Is a Dominant Remodeler of the Effector T Cell Surface Proteome Relative to Activation and Regulatory T Cell Suppression.

Immunosuppressive factors in the tumor microenvironment (TME) impair T cell function and limit the antitumor immune response. T cell surface receptors and surface proteins that influence interactions and function in the TME are proven targets for cancer immunotherapy. However, how the entire surface proteome remodels in primary human T cells in response to specific suppressive factors in the TME remains to be broadly and systematically characterized. Here, using a reductionist cell culture approach with primary human T cells and stable isotopic labeling with amino acids in cell culture-based quantitative cell surface capture glycoproteomics, we examined how two immunosuppressive TME factors, regulatory T cells (Tregs) and hypoxia, globally affect the activated CD8+ surface proteome (surfaceome). Surprisingly, coculturing primary CD8+ T cells with Tregs only modestly affected the CD8+ surfaceome but did partially reverse activation-induced surfaceomic changes. In contrast, hypoxia drastically altered the CD8+ surfaceome in a manner consistent with both metabolic reprogramming and induction of an immunosuppressed state. The CD4+ T cell surfaceome similarly responded to hypoxia, revealing a common hypoxia-induced surface receptor program. Our surfaceomics findings suggest that hypoxic environments create a challenge for T cell activation. These studies provide global insight into how Tregs and hypoxia remodel the T cell surfaceome and we believe represent a valuable resource to inform future therapeutic efforts to enhance T cell function.

Phase I prospective trial of TAS-102 (trifluridine and tipiracil) and radioembolization with 90Y resin microspheres for chemo-refractory colorectal liver metastases.

BACKGROUND: Extrahepatic disease progression limits clinical efficacy of Yttrium-90 (90Y) radioembolization (TARE) for patients with chemotherapy-refractory metastatic colorectal cancer (mCRC). Trifluridine and tipiracil (TAS-102) has overall survival benefit for patients with refractory mCRC and may be a radiosensitizer. METHODS: Sequential lobar TARE using 90Y resin microspheres in combination with TAS-102 in 28-day cycles were used to treat adult patients with bilobar liver-dominant chemo-refractory mCRC according to 3 + 3 dose escalation design with a 12-patient dose expansion cohort. Study objectives were to establish safety and determine maximum tolerated dose (MTD) of TAS-102 in combination with TARE. RESULTS: A total of 21 patients (14 women, 7 men) with median age of 60 years were enrolled. No dose limiting toxicities were observed. Treatment related severe adverse events included cytopenias (10 patients, 48%) and radioembolization-induced liver disease (2 patients, 10%). Disease control rate in the liver lobes treated with TARE was 100%. Best observed radiographic responses were partial response for 4 patients (19%) and stable disease for 12 patients (57%). CONCLUSIONS: The combination of TAS-102 and TARE for patients with liver-dominant mCRC is safe and consistently achieves disease control within the liver. TRIAL REGISTRATION: ClinicalTrials.gov identifier NCT02602327 (first posted 11/11/2015).

Implementation of an Embedded In-Clinic Genetic Testing Station to Optimize Germline Testing for Patients with Pancreatic Adenocarcinoma.

BACKGROUND: Germline genetic testing is universally recommended for patients with pancreatic cancer, but testing remains infrequent. In May 2018, we implemented a systematic patient intake workflow featuring an in-clinic genetic testing station (GTS) at the University of California San Francisco (UCSF) to expedite genetic counseling and facilitate sample collection. We sought to determine the impact of this innovation on rates of genetic counseling and testing. METHODS: Medical records, patient intake records, and genetic test reports were retrospectively reviewed for new patients with pancreatic cancer eligible for germline testing at UCSF from May 2018 to May 2019. Primary outcomes included the rate of offered genetic counseling and confirmed germline testing. Data were compared for periods before and after GTS implementation. Associations between demographic characteristics and testing rates were assessed. RESULTS: Genetic counseling/testing was offered to 209 (94%) of 223 eligible patients, and 158 (71%) completed testing (135 at UCSF, 23 elsewhere). Compared with a traditional referral-based genetic counseling model, confirmed testing increased from 19% to 71%, patient attrition between referral and genetics appointment decreased from 36% to 3%, and rate of pathogenic variant detection increased from 20% to 33%. Patients who were younger, identified as non-Hispanic White, and spoke English as a primary language were more likely to complete testing. CONCLUSIONS: Implementation of a systematic patient intake workflow and in-clinic GTS resulted in the highest reported real-world rate of germline testing for patients with pancreatic cancer. Health care disparities were identified and will guide future innovation. This report provides a model for other centers to create a similar testing infrastructure. IMPLICATIONS FOR PRACTICE: This study demonstrates that a systematic patient intake workflow and associated in-clinic genetic testing station improve delivery of genetic counseling and completion of germline testing for patients with pancreatic cancer. This study achieved, to the authors' knowledge, the highest real-world rate of confirmed genetic testing in this patient population. This article describes this innovation in detail to guide replication at other medical centers and facilitate guideline-concordant care for patients with pancreatic cancer. This infrastructure can also be applied to other cancers for which germline testing is recommended.

MM-398 (nanoliposomal irinotecan): emergence of a novel therapy for the treatment of advanced pancreatic cancer.

While progress in the treatment of advanced pancreatic cancer has accelerated in recent years, this malignancy continues to have an exceedingly poor prognosis, with no standard of care options beyond front-line chemotherapy. Currently, there are a number of new therapeutic agents in varying stages of clinical development, including molecularly targeted agents, immunotherapies, and modified versions of cytotoxics. MM-398, a novel nanoliposomal formulation of irinotecan, was designed to maximize tumor exposure while minimizing systemic toxicity due to its favorable pharmacokinetic profile. Overall, across multiple clinical trials in multiple disease indications, MM-398 has been shown to have a favorable safety and tolerability profile compared with standard irinotecan. Recent results of the Phase III NAPOLI-1 trial in patients with metastatic pancreatic cancer refractory to gemcitabine-based chemotherapy have shown a significant improvement in overall survival of MM-398 when combined with 5-fluorouracil/leucovorin, compared with 5-fluorouracil/leucovorin alone. This review focuses on the development and pharmacokinetic properties of MM-398, followed by evaluation of its safety and efficacy with a primary emphasis on clinical trials in advanced pancreatic cancer.

Progress in central nervous system lymphomas.

Until recently, primary central nervous system lymphoma (PCNSL) was associated with a uniformly dismal prognosis. It is now reasonable to anticipate long-term survival and possibly cure for a significant proportion of patients diagnosed with PCNSL. Accumulated data generated over the past 10 years has provided evidence that long-term progression-free survival (PFS) can reproducibly be attained in a significant fraction of PCNSL patients that receive dose-intensive chemotherapy consolidation, without whole brain radiotherapy. One consolidative regimen that has reproducibly demonstrated promise is the combination of infusional etoposide plus high-dose cytarabine (EA), administered in first complete remission after methotrexate, temozolomide and rituximab-based induction. Given evolving principles of management and the mounting evidence for reproducible improvements in survival rates in prospective clinical series, our goal in this review is to highlight and update principles in diagnosis, staging and management as well as to review data regarding the pathogenesis of central nervous system lymphomas, information that is likely to constitute a basis for the implementation of novel therapies that are requisite for further progress in this unique phenotype of non-Hodgkin lymphoma.

The Additional Diagnostic Benefit of Pancreatic Cancer Molecular Profiling After Germline Testing.

OBJECTIVES: Germline genetic testing is universally recommended for patients with pancreatic cancer to guide therapeutic selection, but tumor molecular profiling (TMP) is not. We aimed to determine the real-world additional diagnostic benefit of TMP after germline testing for detecting therapeutically actionable alterations. METHODS: Medical records and genetic test reports were reviewed for all patients who underwent germline testing and TMP at the University of California San Francisco during January 2016-January 2020. The detection rate of actionable alterations with germline testing alone was compared to that with both germline testing and TMP. RESULTS: Among 738 eligible patients, 144 (20%) met study criteria. Germline testing detected 10 actionable alterations in 10 patients. Tumor molecular profiling identified 3 new therapeutic targets among these 10 patients and 45 targets in 41 additional patients, increasing the number of patients with actionable findings from 10 (7%) to 51 (35%). Most actionable alterations (35/58, 60%) involved genes associated with the Homologous Recombination DNA Damage Repair pathway. CONCLUSIONS: Tumor molecular profiling after germline testing increased the detection of actionable alterations by 5-fold. Tumor molecular profiling is a necessary complement to germline genetic testing to fully inform therapeutic decision making for all patients with pancreatic cancer.

Resistance to ATR Inhibitors Is Mediated by Loss of the Nonsense-Mediated Decay Factor UPF2.

Over one million cases of gastric cancer are diagnosed each year globally, and the metastatic disease continues to have a poor prognosis. A significant proportion of gastric tumors have defects in the DNA damage response pathway, creating therapeutic opportunities through synthetic lethal approaches. Several small-molecule inhibitors of ATR, a key regulator of the DNA damage response, are now in clinical development as targeted agents for gastric cancer. Here, we performed a large-scale CRISPR interference screen to discover genetic determinants of response and resistance to ATR inhibitors (ATRi) in gastric cancer cells. Among the top hits identified as mediators of ATRi response were UPF2 and other components of the nonsense-mediated decay (NMD) pathway. Loss of UPF2 caused ATRi resistance across multiple gastric cancer cell lines. Global proteomic, phosphoproteomic, and transcriptional profiling experiments revealed that cell-cycle progression and DNA damage responses were altered in UPF2-mutant cells. Further studies demonstrated that UPF2-depleted cells failed to accumulate in G1 following treatment with ATRi. UPF2 loss also reduced transcription-replication collisions, which has previously been associated with ATRi response, thereby suggesting a possible mechanism of resistance. Our results uncover a novel role for NMD factors in modulating response to ATRi in gastric cancer, highlighting a previously unknown mechanism of resistance that may inform the clinical use of these drugs. SIGNIFICANCE: Loss of NMD proteins promotes resistance to ATR inhibitors in gastric cancer cells, which may provide a combination of therapeutic targets and biomarkers to improve the clinical utility of these drugs.

Genome-wide CRISPR screens of T cell exhaustion identify chromatin remodeling factors that limit T cell persistence.

T cell exhaustion limits antitumor immunity, but the molecular determinants of this process remain poorly understood. Using a chronic stimulation assay, we performed genome-wide CRISPR-Cas9 screens to systematically discover regulators of T cell exhaustion, which identified an enrichment of epigenetic factors. In vivo CRISPR screens in murine and human tumor models demonstrated that perturbation of the INO80 and BAF chromatin remodeling complexes improved T cell persistence in tumors. In vivo Perturb-seq revealed distinct transcriptional roles of each complex and that depletion of canonical BAF complex members, including Arid1a, resulted in the maintenance of an effector program and downregulation of exhaustion-related genes in tumor-infiltrating T cells. Finally, Arid1a depletion limited the acquisition of exhaustion-associated chromatin accessibility and led to improved antitumor immunity. In summary, we provide an atlas of the genetic regulators of T cell exhaustion and demonstrate that modulation of epigenetic state can improve T cell responses in cancer immunotherapy.

Knowledge base and caretaker-child communication about HIV in peri-urban schoolchildren of Lusaka, Zambia.

Peri-urban communities, which face health risks of both urban and rural environments, have grown extensively with recent global urbanization. These communities' combination of multiple HIV risk factors with the lack of a formalized sexual education system sets the stage for high-risk behavior in peri-urban youth. We conducted a cross-sectional survey of children (ages 5-17, N=331) and accompanying caretakers in peri-urban Lusaka, Zambia, using both closed- and open-ended questions to investigate HIV knowledge and communication. We found that while 67% of children had heard of HIV, only 26% and 23% could accurately name a major mode of transmission and prevention, respectively. In a multivariate model, increasing age was the only significant demographic correlate of a child's ability to offer accurate responses to either question. Though HIV/AIDS knowledge levels were high in the eldest (14-17-year old) age group, in the 10-13-year-old age group accurate modes of transmission and prevention were provided by only 41% and 33% of study participants, respectively. Sharp instruments, particularly razor blades, were mentioned nearly as frequently as sexual intercourse with respect to both transmission and prevention, a response trend that predominated in the youngest age group but persisted into the oldest. Seventy percent of caretakers had not spoken with their child about HIV. A history of caretaker-child communication about HIV was associated with an increased likelihood of a child offering an accurate mode of transmission (OR 2.70, 95% confidence interval (CI) 1.41-5.18, p=0.0029) or prevention (OR 3.43, 95% CI 1.78-6.60, p=0.0002), in multivariate analyses. Our results demonstrate a lack of knowledge and dialog about HIV in the pre-adolescent subset of high-risk peri-urban youth, and uncover undue emphasis on razor blades as a major mode of disease transmission.

Durable response after immune checkpoint inhibitor-related diabetes in mismatch repair-deficient pancreatic cancer.

Mismatch repair protein deficiency occurs in 0.8-2% of pancreatic ductal adenocarcinomas and confers susceptibility to immunotherapy. Herein, we report the case of a patient with Lynch syndrome-associated, locally advanced mismatch repair protein deficiency pancreatic ductal adenocarcinomas who demonstrated a sustained response to second-line treatment with pembrolizumab, but eventually developed immune-related diabetic ketoacidosis requiring discontinuation of treatment. He has since remained in remission, off treatment, over the following 3 years, with regular surveillance showing no clinical or radiographic evidence of disease progression. The patient's unusual disease course raises the question of whether this serious immune-related adverse event affecting the organ of malignant involvement may have predicted his remarkable and durable response.

Lay abstract A small subgroup of pancreatic cancers have mutations preventing effective repair of damaged DNA; a condition termed 'mismatch repair protein deficiency'. These tumors are often effectively treated with immunotherapy. Here we describe a patient whose mismatch repair protein deficiency pancreatic cancer responded well to pembrolizumab immunotherapy, but who later developed diabetes as an immunotherapy-related adverse effect. Treatment was stopped, but his tumor remained stable off treatment over the next 3 years. His unique clinical course raises the question of whether the development of diabetes, a pancreas-specific adverse effect, may have predicted the effective treatment of pancreatic cancer.

Schistosomiasis haematobium prevalence and risk factors in a school-age population of peri-urban Lusaka, Zambia.

Given association of the parasite Schistosoma haematobium with coastal and rural/agricultural populations, there is little documentation to date of infection patterns in today's rapidly urbanizing non-coastal regions. We conducted an observational study of 5-17-year-old school children (N = 1583) in peri-urban compounds of Lusaka, Zambia. Demographic information, medical history, physical examination findings and urinalysis results were recorded. Prevalence of schistosomiasis in the population was 20.72%. Significant risk factors for infection were male gender [odds ratio (OR) 2.42], age of 9-12 years or 13-17 years (OR 3.33 and 3.26, respectively, compared with 5-8-year-olds) and single and/or double orphan status (OR 1.43). Clinical officers detected schistosomiasis with a sensitivity of 24.70% and a specificity of 98.17% after history and physical examination. These results reveal that peri-urban populations have a significant but under-recognized vulnerability to infection, and suggest that only history and physical examination are inadequate for identifying a treatment population.

Referral frequency, attrition rate, and outcomes of germline testing in patients with pancreatic adenocarcinoma.

Hereditary predisposition is estimated to account for 10% of all pancreatic cancer cases. However, referral patterns and clinical workflow for germline testing in this disease differ significantly by institution, and many at-risk patients may not undergo appropriate counseling and testing. We undertook an analysis of patients diagnosed with pancreatic cancer (PDAC) who were referred to the Clinical Genetics program of a high-volume academic center over a 3-year period to assess referral frequency, evaluate the yield of germline testing in this selected patient cohort, and elucidate the reasons individuals did not undergo recommended germline testing. Medical records of patients with PDAC referred for genetic counseling between January 2015 and October 2017 were reviewed for demographic, medical/family history, and disease-specific data. If testing did not occur, reasons were documented. Genetic test results were categorized as negative, variants of unknown significance, or established pathogenic mutations. Descriptive statistics included means with standard deviations; associations were analyzed with t test and Fisher's exact test. 32% (137 of 432) of PDAC patients were referred for genetic counseling, but only 64% attended their appointment and 60% ultimately underwent germline testing. Common reasons for attrition included worsening disease severity, lack of patient follow-up, insurance concerns, and logistic/travel challenges. Pathogenic germline mutations were detected in 20% (16 of 82) of patients tested, distributed across races/ethnicities, and significantly associated with younger age and positive family history of breast cancer. PDAC patients frequently do not undergo genetic counseling/germline testing despite appropriate referrals, highlighting a need to develop streamlined processes to engage more patients in testing, especially those with high-risk features.

The Challenge of Primary Central Nervous System Lymphoma.

Primary central nervous system (CNS) lymphoma is a challenging subtypes of aggressive non-Hodgkin lymphoma. Emerging clinical data suggest that optimized outcomes are achieved with dose-intensive CNS-penetrant chemotherapy and avoiding whole brain radiotherapy. Anti-CD20 antibody-based immunotherapy as a component of high-dose methotrexate-based induction programs may contribute to improved outcomes. An accumulation of insights into the molecular and cellular basis of disease pathogenesis is providing a foundation for the generation of molecular tools to facilitate diagnosis as well as a roadmap for integration of targeted therapy within the developing therapeutic armamentarium for this challenging brain tumor.

The crimson care collaborative: a student-faculty initiative to increase medical students' early exposure to primary care.

The current shortage of primary care physicians (PCPs), particularly as more individuals obtain health insurance and seek primary care services, is a growing national concern. The Crimson Care Collaborative (CCC) is a joint student-faculty initiative in post-health-care-reform Massachusetts that was started with the explicit goal of attracting medical students to primary care careers. It fills a niche for student-run clinics, providing evening access to primary care services for patients without a PCP and urgent care services for patients of a Massachusetts General Hospital-affiliated internal medicine clinic, with the aim of decreasing emergency department use in both groups. Unlike other student-run clinics, CCC is integrated into the mainstream health care structure of an existing primary care clinic and, because of universal health insurance coverage in Massachusetts, can bill for its services. In addition to the clinical services offered, the student-run research team evaluates the quality of care and the patients' experiences at the clinic. This article describes the creation and development of CCC, including a brief overview of clinic operations, social services, research, laboratory services, student and patient education programs, and finance. In the wake of the Patient Protection and Affordable Care Act of 2010, CCC is an example of how students can aid the transition to universal health care in the United States and how medical schools can expose students early in their training to primary care and clinic operations.

The development of sentence interpretation: effects of perceptual, attentional and semantic interference.

How does the development and consolidation of perceptual, attentional, and higher cognitive abilities interact with language acquisition and processing? We explored children's (ages 5-17) and adults' (ages 18-51) comprehension of morphosyntactically varied sentences under several competing speech conditions that varied in the degree of attentional demands, auditory masking, and semantic interference. We also evaluated the relationship between subjects' syntactic comprehension and their word reading efficiency and general 'speed of processing'. We found that the interactions between perceptual and attentional processes and complex sentence interpretation changed considerably over the course of development. Perceptual masking of the speech signal had an early and lasting impact on comprehension, particularly for more complex sentence structures. In contrast, increased attentional demand in the absence of energetic auditory masking primarily affected younger children's comprehension of difficult sentence types. Finally, the predictability of syntactic comprehension abilities by external measures of development and expertise is contingent upon the perceptual, attentional, and semantic milieu in which language processing takes place.