Implementation of a gene therapy education initiative by the ASGCT and Muhimbili University of Health and Allied Sciences.

There has been rapid growth in gene therapy development with an expanding list of approved clinical products. Several therapies are particularly relevant to patients in low- and middle-income countries. Moreover, investing in research and manufacturing presents an opportunity for economic development. To increase awareness of gene therapy, the American Society of Gene and Cell Therapy partnered with the Muhimbili University of Health and Allied Sciences, Tanzania, to create a certificate-bearing course. The goal was to provide faculty teaching in graduate and medical schools with the tools needed to add gene therapy to the university curriculum. The first virtual course was held in October of 2022, and 45 individuals from 9 countries in Africa completed the training. The content was new to approximately two-thirds of participants, with the remaining third indicating that the course increased their knowledge base. The program was well received and will be adapted for other under-resourced regions.

Ablation of the endoplasmic reticulum stress kinase PERK induces paraptosis and type I interferon to promote anti-tumor T cell responses.

Activation of unfolded protein responses (UPRs) in cancer cells undergoing endoplasmic reticulum (ER) stress promotes survival. However, how UPR in tumor cells impacts anti-tumor immune responses remains poorly described. Here, we investigate the role of the UPR mediator pancreatic ER kinase (PKR)-like ER kinase (PERK) in cancer cells in the modulation of anti-tumor immunity. Deletion of PERK in cancer cells or pharmacological inhibition of PERK in melanoma-bearing mice incites robust activation of anti-tumor T cell immunity and attenuates tumor growth. PERK elimination in ER-stressed malignant cells triggers SEC61ß-induced paraptosis, thereby promoting immunogenic cell death (ICD) and systemic anti-tumor responses. ICD induction in PERK-ablated tumors stimulates type I interferon production in dendritic cells (DCs), which primes CCR2-dependent tumor trafficking of common-monocytic precursors and their intra-tumor commitment into monocytic-lineage inflammatory Ly6C+CD103+ DCs. These findings identify how tumor cell-derived PERK promotes immune evasion and highlight the potential of PERK-targeting therapies in cancer immunotherapy.

pH Sensitive Erythrocyte-Derived Membrane for Acute Systemic Retention and Increased Infectivity of Coated Oncolytic Vaccinia Virus.

Oncolytic viruses have emerged as a promising modality in cancer treatment given their high synergy with highly efficient immune checkpoint inhibitors. However, their potency is limited by their rapid in vivo clearance. To overcome this, we coated oncolytic vaccinia viruses (oVV) with erythrocyte-derived membranes (EDMs), hypothesizing that they would not only remain in systemic circulation for longer as erythrocytes would when administered intravenously, but also respond to environmental pH cues due to their membrane surface sialic acid residues. For this, we developed a model based on DLVO theory to show that the acidic moieties on the surface of EDM confers it the ability to respond to pH-based stimuli. We corroborate our modeling results through in vitro cell culture models and show that EDM-coated oVV infects cancer cells faster under acidic conditions akin to the tumor microenvironment. When EDM-coated oVVs were intravenously injected into wild-type mice, they exhibited prolonged circulation at higher concentrations when compared to the unprocessed oVV. Furthermore, when EDM-coated oVV was directly injected into xenografted tumors, we observed that they were suppressed earlier than the tumors that received regular oVV, suggesting that the EDM coating does not hinder oVV infectivity. Overall, we found that EDM was able to serve as a multi-functional encapsulant that allowed the payload to remain in circulation at higher concentrations when administered intravenously while simultaneously exhibiting pH-responsive properties.

Tcf-1 protects anti-tumor TCR-engineered CD8+ T-cells from GzmB mediated self-destruction.

BACKGROUND: T-cell longevity is undermined by antigen-driven differentiation programs that render cells prone to attrition through several mechanisms. CD8 + T cells that express the Tcf-1 transcription factor have undergone limited differentiation and exhibit stem-cell-like replenishment functions that facilitate persistence. We engineered human CD8 + T cells to constitutively express Tcf-1 and a TCR specific for the NY-ESO-1 cancer-associated antigen. Co-engineered cells were assessed for their potential for adoptive cellular immunotherapy. METHODS: Tcf-1 mRNA encoding TCF-1B and TCF-1E isoforms, along with GzmB expression were assessed in CD62L + CD57 -, CD62L - CD57 -, and CD62L - CD57 + CD8 + T cells derived from normal donor lymphocytes. The impact of stable Tcf-1B expression on CD8 + T-cell phenotype, anti-tumor activity, and cell-cycle activity was assessed in vitro and in an in vivo tumor xenograft model. RESULTS: TCF-1B and TCF-1E were dynamically regulated during self-renewal, with progeny of recently activated naïve T cells more enriched for TCF-1B mRNA. Constitutive TCF-1B expression improved the survival of TCR-engineered CD8 + T cells upon engagement with tumor cells. Tcf-1B prohibited the acquisition of a GzmB High state, and protected T cells from apoptosis associated with elicitation of effector function, and promoted stem cell-like characteristics. CONCLUSIONS: Tcf-1 protects TCR-engineered CD8 + T cells from activation induced cell death by restricting GzmB expression. Our study presents constitutive Tcf-1B expression as a potential means to impart therapeutic T cells with attributes of persistence for durable anti-tumor activity.

Metabolic adaptation of ovarian tumors in patients treated with an IDO1 inhibitor constrains antitumor immune responses.

To uncover underlying mechanisms associated with failure of indoleamine 2,3-dioxygenase 1 (IDO1) blockade in clinical trials, we conducted a pilot, window-of-opportunity clinical study in 17 patients with newly diagnosed advanced high-grade serous ovarian cancer before their standard tumor debulking surgery. Patients were treated with the IDO1 inhibitor epacadostat, and immunologic, transcriptomic, and metabolomic characterization of the tumor microenvironment was undertaken in baseline and posttreatment tumor biopsies. IDO1 inhibition resulted in efficient blockade of the kynurenine pathway of tryptophan degradation and was accompanied by a metabolic adaptation that shunted tryptophan catabolism toward the serotonin pathway. This resulted in elevated nicotinamide adenine dinucleotide (NAD+), which reduced T cell proliferation and function. Because NAD+ metabolites could be ligands for purinergic receptors, we investigated the impact of blocking purinergic receptors in the presence or absence of NAD+ on T cell proliferation and function in our mouse model. We demonstrated that A2a and A2b purinergic receptor antagonists, SCH58261 or PSB1115, respectively, rescued NAD+-mediated suppression of T cell proliferation and function. Combining IDO1 inhibition and A2a/A2b receptor blockade improved survival and boosted the antitumor immune signature in mice with IDO1 overexpressing ovarian cancer. These findings elucidate the downstream adaptive metabolic consequences of IDO1 blockade in ovarian cancers that may undermine antitumor T cell responses in the tumor microenvironment.

Engineering tumor stromal mechanics for improved T cell therapy.

Adoptive cellular therapies (ACT), including the engineered T cell receptor (TCR) therapy and chimeric antigen receptor (CAR) T Cell Therapy, are currently at the forefront of cancer immunotherapy. However, their efficacy for the treatment of solid tumors has not been confirmed. The fibrotic stroma surrounding the solid tumor has been suggested as the main barrier in the disarmament and suppression of the engineered T cells. In this review, we will discuss the recent findings on the mechanism of T cell suppression by the tumor stroma with a special emphasis on the effect of stromal mechanics. We will also discuss the engineering approaches used to dissect the mechanism of the T cell suppression by the stromal mechanical factors. Finally, we will provide a future outlook on the strategies to improve the efficacy of T cell therapy through altering the tumor stromal fibrosis.

CXCR6 by increasing retention of memory CD8+ T cells in the ovarian tumor microenvironment promotes immunosurveillance and control of ovarian cancer.

PURPOSE: Resident memory CD8 T cells, owing to their ability to reside and persist in peripheral tissues, impart adaptive sentinel activity and amplify local immune response, and have beneficial implications for tumor surveillance and control. The current study aimed to clarify the less known chemotactic mechanisms that govern the localization, retention, and residency of memory CD8 T cells in the ovarian tumor microenvironment. EXPERIMENTAL DESIGN: RNA and protein expressions of chemokine receptors in CD8+ resident memory T cells in human ovarian tumor-infiltrating CD8+ T cells and their association with survival were analyzed. The role of CXCR6 on antitumor T cells was investigated using prophylactic vaccine models in murine ovarian cancer. RESULTS: Chemokine receptor profiling of CD8+CD103+ resident memory tumor-infiltrating lymphocytes in patients with ovarian cancer revealed high expression of CXCR6. Analysis of The Cancer Genome Atlas (TCGA) (ovarian cancer database revealed CXCR6 to be associated with CD103 and increased patient survival. Functional studies in mouse models of ovarian cancer revealed that CXCR6 is a marker of resident, but not circulatory, tumor-specific memory CD8+ T cells. CXCR6-deficient tumor-specific CD8+ T cells showed reduced retention in tumor tissues, leading to diminished resident memory responses and poor control of ovarian cancer. CONCLUSIONS: CXCR6, by promoting retention in tumor tissues, serves a critical role in resident memory T cell-mediated immunosurveillance and control of ovarian cancer. Future studies warrant exploiting CXCR6 to promote resident memory responses in cancers.

Engineered microenvironment for the study of myofibroblast mechanobiology.

Myofibroblasts are mechanosensitive cells and a variety of their behaviours including differentiation, migration, force production and biosynthesis are regulated by the surrounding microenvironment. Engineered cell culture models have been developed to examine the effect of microenvironmental factors such as the substrate stiffness, the topography and strain of the extracellular matrix (ECM) and the shear stress on myofibroblast biology. These engineered models provide well-mimicked, pathophysiologically relevant experimental conditions that are superior to those enabled by the conventional two-dimensional (2D) culture models. In this perspective, we will review the recent advances in the development of engineered cell culture models for myofibroblasts and outline the findings on the myofibroblast mechanobiology under various microenvironmental conditions. These studies have demonstrated the power and utility of the engineered models for the study of microenvironment-regulated cellular behaviours. The findings derived using these models contribute to a greater understanding of how myofibroblast behaviour is regulated in tissue repair and pathological scar formation.

WHSC1/NSD2 regulates immune infiltration in prostate cancer.

BACKGROUND: Immunotherapy in prostate cancer (PCa) lags behind the progresses obtained in other cancer types partially because of its limited immune infiltration. Tumor-resident immune cells have been detected in the prostate, but the regulatory mechanisms that govern tumor infiltration are still poorly understood. To address this gap, we investigated the role of Wolf-Hirschhorn syndrome candidate 1 (WHSC1), a histone methyltransferase enzyme that targets dimethyl and trimethyl H3K36. WHSC1 is known to promote malignant growth and progression in multiple tumors, but its role in the interface between PCa and immune system is unknown. METHODS: RNA Sequencing (RNASeq) data from patients with PCa from The Cancer Genome Atlas (TCGA) were collected and divided into top/bottom 30% based on the expression of WHSC1 and disease-free survival was calculated. Publicly available chromatin immunoprecipitation (ChIPSeq) data were obtained from Cistrome and integrated with the available RNASeq data. RNASeq, ATACSeq and methylomic were analyzed using R Bioconductor packages comparing C42 cells with or without stable knockdown on WHSC1. Flow cytometry was used to measure Major Histocompatibility complex (MHC) levels, MHC-bound ovalbumin and tumor infiltration. C57B6 and NOD scid gamma (NSG) mice were subcutaneously grafted with TRansgenic Adenocarcinoma of the Mouse Prostate (TRAMP) C2 cells and treated with MCTP39 (10 mg/kg); tumor size was monitored over time and curves were compared using permutation analyses. All analyses used a significance threshold of 0.05. RESULTS: Leveraging TCGA data, we demonstrated that elevated WHSC1 levels positively correlate with the presence of an immunosuppressive microenvironment. We validated those results in vitro, demonstrating that genetic and pharmacological inhibition of WHSC1 restores antigen presentation. This occurs via an elegant epigenetic regulation of gene expression at the chromatin and DNA methylation levels. In vivo studies in immunocompetent mice also show an increased frequency of CD8+ T cells in tumors from mice treated with WHSC1 inhibitor, supporting the hypothesis that the antitumor effect following WHSC1 inhibition requires a fully functional immune system. CONCLUSIONS: This study demonstrates a novel role for WHSC1 in defining immune infiltration in PCa, with significant future implications for the use of immunotherapies in prostate malignancies.

Synthesis, Tumor Specificity, and Photosensitizing Efficacy of Erlotinib-Conjugated Chlorins and Bacteriochlorins: Identification of a Highly Effective Candidate for Photodynamic Therapy of Cancer.

Erlotinib was covalently linked to 3-(1'-hexyloxy)ethyl-3-devinylpyropheophorbide-a (HPPH) and structurally related chlorins and bacteriochlorins at different positions of the tetrapyrrole ring. The functional consequence of each modification was determined by quantifying the uptake and subcellular deposition of the erlotinib conjugates, cellular response to therapeutic light treatment in tissue cultures, and in eliminating of corresponding tumors grown as a xenograft in SCID mice. The experimental human cancer models the established cell lines UMUC3 (bladder), FaDu (hypopharynx), and primary cultures of head and neck tumor cells. The effectiveness of the compounds was compared to that of HPPH. Furthermore, specific functional contribution of the carboxylic acid side group at position 172 and the chiral methyl group at 3(1') to the overall activity of the chimeric compounds was assessed. Among the conjugates investigated, the PS 10 was identified as the most effective candidate for achieving tumor cell-specific accumulation and yielding improved long-term tumor control.

Cellular Immunotherapy in the Treatment of Hematopoietic Malignancies.

Cancer immunotherapy has been shown to be an efficacious therapeutic approach in the treatment of cancers including hematopoietic malignancies. Induction of T cell cytotoxicity against tumors by adoptive cell therapies (ACT), cancer vaccines, gene therapies, and monoclonal antibody therapies has been intensively studied. In particular, immune checkpoint blockade and chimeric antigen receptor T (CAR-T) cell therapies are the recent clinical successes in cancer immunotherapy. This article introduces the main concepts and addresses the most relevant clinical modalities of cellular immunotherapies for hematological malignancies: antigen non-specific T cell therapy, genetically modified T cell receptor (TCR) T cell therapy, chimeric antigen receptor (CAR) T cell therapy, and CAR-T cell clinical trials in leukemia, lymphoma, and multiple myeloma. Clinical trials have shown encouraging results, but future studies may need to incorporate novel CAR constructs or targets with enhanced safety and efficacy to ensure long-term benefits.

Efficient identification of neoantigen-specific T-cell responses in advanced human ovarian cancer.

BACKGROUND: Efficient identification of neoantigen-specific T-cell responses in epithelial ovarian cancer (EOC) remains a challenge. Existing investigations of spontaneous T-cell response to tumor neoepitope in EOC have taken the approach of comprehensive screening all neoantigen candidates, with a validation rate of 0.5-2%. METHODS: Whole-exome and transcriptome sequencing analysis of treatment-naive EOC patients were performed to identify neoantigen candidates, and the immunogenicity of prioritized neoantigens was evaluated by analyzing spontaneous neoantigen-specfic CD4+ and CD8+ T-cell responses in the tumor and/or peripheral blood. The biological relevance of neoantigen-specific T-cell lines and clones were analyzed by evaluating the capacity of autologous ovarian tumor recognition. Genetic transfer of T-cell receptor (TCR) from these neoantigen-specific T-cell clones into peripheral blood T-cells was conducted to generate neoepitope-specific T-cells. The molecular signature associated with positive neoantigen T-cell responses was investigated, and the impacts of expression level and lymphocyte source on neoantigen identification were explored. RESULTS: Using a small subset of prioritized neoantigen candidates, we were able to detect spontaneous CD4+ and/or CD8+ T-cell responses against neoepitopes from autologous lymphocytes in half of treatment-naïve EOC patients, with a significantly improved validation rate of 19%. Tumors from patients exhibiting neoantigen-specific T-cell responses exhibited a signature of upregulated antigen processing and presentation machinery, which was also associated with favorable patient survival in the TCGA ovarian cohort. T-cells specific against two mutated cancer-associated genes, NUP214 and JAK1, recognized autologous tumors. Gene-engineering with TCR from these neoantigen-specific T-cell clones conferred neoantigen-reactivity to peripheral T-cells. CONCLUSIONS: Our study demonstrated the feasibility of efficiently identifying both CD4+ and CD8+ neoantigen-specific T-cells in EOC. Autologous lymphocytes genetically engineered with tumor antigen-specific TCR can be used to generate cells for use in the personalized adoptive T-cell transfer immunotherapy.

Neoantigens retention in patient derived xenograft models mediates autologous T cells activation in ovarian cancer.

Ovarian cancer (OC) has an overall modest number of mutations that facilitate a functional immune infiltrate able to recognize tumor mutated antigens, or neoantigens. Although patient-derived xenografts (PDXs) can partially model the tumor mutational load and mimic response to chemotherapy, no study profiled a neoantigen-driven response in OC PDXs. Here we demonstrate that the genomic status of the primary tumor from an OC patient can be recapitulated in vivo in a PDX model, with the goal of defining autologous T cells activation by neoantigens using in silico, in vitro and in vivo approaches. By profiling the PDX mutanome we discovered three main clusters of mutations defining the expansion, retraction or conservation of tumor clones based on their variant allele frequencies (VAF). RNASeq analyses revealed a strong functional conservation between the primary tumor and PDXs, highlighted by the upregulation of antigen presenting pathways. We tested in vitro a set of 30 neoantigens for recognition by autologous T cells and identified a core of six neoantigens that define a potent T cell activation able to slow tumor growth in vivo. The pattern of recognition of these six neoantigens indicates the pre-existence of anti-tumor immunity in the patient. To evaluate the breadth of T cell activation, we performed single cell sequencing profiling the TCR repertoire upon stimulation with neoantigenic moieties and identified sequence motifs that define an oligoclonal and autologous T cell response. Overall, these results indicate that OC PDXs can be a valid tool to model OC response to immunotherapy.

Mature neutrophils suppress T cell immunity in ovarian cancer microenvironment.

Epithelial ovarian cancer (EOC) often presents with metastases and ascites. Granulocytic myeloid-derived suppressor cells are an immature population that impairs antitumor immunity. Since suppressive granulocytes in the ascites of patients with newly diagnosed EOC were morphologically mature, we hypothesized that PMN were rendered suppressive in the tumor microenvironment (TME). Circulating PMN from patients were not suppressive but acquired a suppressor phenotype (defined as ≥1 log10 reduction of anti-CD3/CD28-stimulated T cell proliferation) after ascites supernatant exposure. Ascites supernatants (20 of 31 supernatants) recapitulated the suppressor phenotype in PMN from healthy donors. T cell proliferation was restored with ascites removal and restimulation. PMN suppressors also inhibited T cell activation and cytokine production. PMN suppressors completely suppressed proliferation in naive, central memory, and effector memory T cells and in engineered tumor antigen-specific cytotoxic T lymphocytes, while antigen-specific cell lysis was unaffected. Inhibition of complement C3 activation and PMN effector functions, including CR3 signaling, protein synthesis, and vesicular trafficking, abrogated the PMN suppressor phenotype. Moreover, malignant effusions from patients with various metastatic cancers also induced the C3-dependent PMN suppressor phenotype. These results point to PMN impairing T cell expansion and activation in the TME and the potential for complement inhibition to abrogate this barrier to antitumor immunity.

A rare population of tumor antigen-specific CD4+CD8+ double-positive αß T lymphocytes uniquely provide CD8-independent TCR genes for engineering therapeutic T cells.

BACKGROUND: High-affinity tumor antigen-specific T-cell receptor (TCR) gene is required to engineer potent T cells for therapeutic treatment of cancer patients. However, discovery of suitable therapeutic TCR genes is hampered by the fact that naturally occurring tumor antigen-specific TCRs are generally of low-affinity, and artificial modification of TCRs can mediate cross-reactivity to other antigens expressed in normal tissues. Here, we discovered a naturally occurring T-cell clone which expressed high-affinity HLA-A*02:01 (A*02)-restricted TCR against NY-ESO-1 from a patient who had NY-ESO-1-expressing ovarian tumor. METHODS: A*02-restricted NY-ESO-1-specific T-cell clones were established from peripheral blood of patients who had NY-ESO-1-expressing ovarian tumors. TCR α and ß chain genes were retrovirally transduced into polyclonally activated T cells. Phenotype and function of the parental and TCR-transduced T cells were analyzed by flow cytometry, ELISA and cytotoxicity assay. In vivo therapeutic efficacy was investigated in a xenograft model using NOD/SCID/IL-2Rγ-deficient (NSG) mice. RESULTS: A rare population of NY-ESO-1-specific T cells, which we named 19305DP, expressed cell surface CD4, CD8α, and CD8ß but not CD56 and recognized A*02+NY-ESO-1+ cancer cell lines in a CD4- and CD8-independent manner. 19305DP showed a gene expression profile that is consistent with a mixed profile of CD4+ and CD8+ single-positive T cells. Both CD4+ and CD8+ T cells that were retrovirally transduced with 19305DP-derived TCR gene (19305DP-TCR) showed strong reactivity against A*02+NY-ESO-1+ cancer cells, whereas TCR genes from the conventional A*02-restricted NY-ESO-1-specific CD8+ single-positive T-cell clones functioned only in CD8+ T cells. Both 19305DP-TCR gene-engineered CD4+ and CD8+ T cells eliminated A*02+NY-ESO-1+ tumor xenografts in NSG mice. Finally, based on reactivity against a series of alanine-substituted peptides and a panel of normal human tissue-derived primary cells, 19305DP-TCR was predicted to have no cross-reactivity against any human non-NY-ESO-1 proteins. CONCLUSION: Together, our results indicate that the naturally occurring 19305DP-TCR derived from CD4+CD8+ double-positive αß T cells, is a promising therapeutic TCR gene for effective and safe adoptive T-cell therapy in A*02+ patients with NY-ESO-1-expressing tumor.

Measurement and Quantitative Characterization of Whole-Body Pharmacokinetics of Exogenously Administered T Cells in Mice.

Here we have investigated whole-body pharmacokinetics (PK) of exogenously administered T cells in a mouse model of melanoma and have developed a physiologically based pharmacokinetic (PBPK) model to quantitatively characterize the data. T cells were isolated from the spleen of tumor-bearing mice, activated, and labeled with chromium-51 to facilitate the quantification. Labeled T cells were injected in the tumor-bearing mice, and PK was measured in 19 different tissues. It was found that T cells disappear from the blood rapidly after administration and accumulate in the tissues to various extents. Spleen, liver, lung, kidney, bone, and lymph nodes accounted for more than 90% of T cells in the body. The distribution of T cells in solid tumors was found to be very low, hovering below 1%ID/g (percent of injected dose per gram of tissue) during the entire study. However, this observation may differ for targeted TCR-T and CAR-T cells. Observed PK profiles also suggest that T-cell-based therapies may be more successful in treating cancers of the lymphatic system and bone marrow metastases compared to solid tumors. A PBPK model was developed to characterize the whole-body PK of T cells, which incorporated key processes such as extravasation, elimination, and recirculation of T cells via lymph flow. Retention factors were incorporated into the spleen, liver, and kidney compartment to adequately capture the PK profiles. The model was able to characterize observed PK profiles reasonably well, and parameters were estimated with good confidence. The PK data and PBPK model presented here provide unprecedented insight into the biodistribution of exogenously administered T cells.

Rapid Construction of Antitumor T-cell Receptor Vectors from Frozen Tumors for Engineered T-cell Therapy.

T cells genetically engineered with tumor antigen-specific T-cell receptor (TCR) genes have demonstrated therapeutic potential in patients with solid tumors. In order to achieve broader application, an efficient method to identify TCR genes for an array of tumor antigens and HLA restriction elements is required. Here, we have developed a method to construct a TCR-expression library from specimens, including frozen tumor biopsies, that contain antigen-specific T cells. TCR-expressing cassettes were constructed and cloned in a retroviral plasmid vector within 24 hours by unbiased PCR amplification of TCR α and ß chain variable regions assembled with TCR constant regions. The method was validated by constructing TCR-expressing vectors from tumor antigen-specific T-cell clones and functionally assessing TCR gene-transduced T cells. We applied this method to frozen ovarian tumor specimens that were infiltrated by tumor antigen-specific T cells. The tumor-derived TCR libraries were expressed in peripheral T cells from healthy volunteers and screened for tumor antigen-specific TCR pairs with the use of an MHC/peptide tetramer reagent. Tumor antigen-specific TCR-expressing transgenes were recovered from isolated tetramer-positive T cells. Peripheral T cells that were engineered with library-derived TCR gene showed potent therapeutic antitumor effect in a tumor xenograft model. Our method can efficiently and rapidly provide tumor-specific TCR-expressing viral vectors for the manufacture of therapeutic and personalized antitumor T-cell products. Cancer Immunol Res; 6(5); 594-604. ©2018 AACR.

Single-cell analysis resolves the cell state transition and signaling dynamics associated with melanoma drug-induced resistance.

Continuous BRAF inhibition of BRAF mutant melanomas triggers a series of cell state changes that lead to therapy resistance and escape from immune control before establishing acquired resistance genetically. We used genome-wide transcriptomics and single-cell phenotyping to explore the response kinetics to BRAF inhibition for a panel of patient-derived BRAFV600 -mutant melanoma cell lines. A subset of plastic cell lines, which followed a trajectory covering multiple known cell state transitions, provided models for more detailed biophysical investigations. Markov modeling revealed that the cell state transitions were reversible and mediated by both Lamarckian induction and nongenetic Darwinian selection of drug-tolerant states. Single-cell functional proteomics revealed activation of certain signaling networks shortly after BRAF inhibition, and before the appearance of drug-resistant phenotypes. Drug targeting those networks, in combination with BRAF inhibition, halted the adaptive transition and led to prolonged growth inhibition in multiple patient-derived cell lines.