FLT1 activation in cancer cells promotes PARP-inhibitor resistance in breast cancer.

Acquired resistance to PARP inhibitors (PARPi) remains a treatment challenge for BRCA1/2-mutant breast cancer that drastically shortens patient survival. Although several resistance mechanisms have been identified, none have been successfully targeted in the clinic. Using new PARPi-resistance models of Brca1- and Bard1-mutant breast cancer generated in-vivo, we identified FLT1 (VEGFR1) as a driver of resistance. Unlike the known role of VEGF signaling in angiogenesis, we demonstrate a novel, non-canonical role for FLT1 signaling that protects cancer cells from PARPi in-vivo through a combination of cell-intrinsic and cell-extrinsic pathways. We demonstrate that FLT1 blockade suppresses AKT activation, increases tumor infiltration of CD8+ T cells, and causes dramatic regression of PARPi-resistant breast tumors in a T-cell-dependent manner. Moreover, PARPi-resistant tumor cells can be readily re-sensitized to PARPi by targeting Flt1 either genetically (Flt1-suppression) or pharmacologically (axitinib). Importantly, a retrospective series of breast cancer patients treated with PARPi demonstrated shorter progression-free survival in cases with FLT1 activation at pre-treatment. Our study therefore identifies FLT1 as a potential therapeutic target in PARPi-resistant, BRCA1/2-mutant breast cancer.

Comparative study of immune response to local tumor destruction modalities in a murine breast cancer model.

Introduction: Immunotherapy is revolutionizing the management of multiple cancer types. However, only a subset of patients responds to immunotherapy. One mechanism of resistance is the absence of immune infiltrates within the tumor. In situ vaccine with local means of tumor destruction that can induce immunogenic cell death have been shown to enhance tumor T cell infiltration and increase efficacy of immune checkpoint blockade. Methods: Here, we compare three different forms of localize tumor destruction therapies: radiation therapy (RT), vascular targeted photodynamic therapy (VTP) and cryoablation (Cryo), which are known to induce immunogenic cell death, with their ability to induce local and systemic immune responses in a mouse 4T1 breast cancer model. The effects of combining RT, VTP, Cryo with anti-PD1 was also assessed. Results: We observed that RT, VTP and Cryo significantly delayed tumor growth and extended overall survival. In addition, they also induced regression of non-treated distant tumors in a bilateral model suggesting a systemic immune response. Flow cytometry showed that VTP and Cryo are associated with a reduction in CD11b+ myeloid cells (granulocytes, monocytes, and macrophages) in tumor and periphery. An increase in CD8+ T cell infiltration into tumors was observed only in the RT group. VTP and Cryo were associated with an increase in CD4+ and CD8+ cells in the periphery. Conclusion: These data suggest that cell death induced by VTP and Cryo elicit similar immune responses that differ from local RT.

Intermittent MEK inhibition with GITR co-stimulation rescues T-cell function for increased efficacy with CTLA-4 blockade in solid tumor models.

MEK inhibitors (MEKis) have shown limited success as a treatment for MAPK/ERK pathway-dependent cancers due to various resistance mechanisms tumor cells can employ. CH5126766 (CKI27) is an inhibitor that binds to MEK and prevents release of RAF, reducing the relief of negative feedback commonly observed with other MEKis. We observed that CKI27 increased MHC expression on tumor cells and improved T cell-mediated killing. Yet, CKI27 also decreased T-cell proliferation, activation, and cytolytic activity by inhibiting the MAPK/ERK pathway that is activated downstream of T cell-receptor signaling. Therefore, we aimed to balance the positive and negative immunomodulatory effects of MEKis for optimal combination with immunotherapy. Intermittent administration of CKI27 allowed T cells to partially recover and co-stimulation via GITR and OX-40 agonist antibodies completely alleviated inhibition of function. In Kras mutant lung and colon tumor mouse models, intermittent CKI27 and anti-GITR significantly decreased tumor growth and prolonged survival when further combined with CTLA-4 immune checkpoint blockade. Moreover, this triple combination increased CD8+ and CD4+ T-cell proliferation, activation, and effector/memory subsets in the tumor draining lymph nodes and tumors and led to intratumoral regulatory T cell (Treg) destabilization. These data, collectively, will allow for more informed decisions when optimizing combination regimens by overcoming resistance, reducing toxicity, and generating long-term immune responses.

TAD hierarchy restricts poised LTR activation and loss of TAD hierarchy promotes LTR co-option in cancer.

Transposable elements (TEs) are abundant in the human genome, and they provide the sources for genetic and functional diversity. The regulation of TEs expression and their functional consequences in physiological conditions and cancer development remain to be fully elucidated. Previous studies suggested TEs are repressed by DNA methylation and chromatin modifications. The effect of 3D chromatin topology on TE regulation remains elusive. Here, by integrating transcriptome and 3D genome architecture studies, we showed that haploinsufficient loss of NIPBL selectively activates alternative promoters at the long terminal repeats (LTRs) of the TE subclasses. This activation occurs through the reorganization of topologically associating domain (TAD) hierarchical structures and recruitment of proximal enhancers. These observations indicate that TAD hierarchy restricts transcriptional activation of LTRs that already possess open chromatin features. In cancer, perturbation of the hierarchical chromatin topology can lead to co-option of LTRs as functional alternative promoters in a context-dependent manner and drive aberrant transcriptional activation of novel oncogenes and other divergent transcripts. These data uncovered a new layer of regulatory mechanism of TE expression beyond DNA and chromatin modification in human genome. They also posit the TAD hierarchy dysregulation as a novel mechanism for alternative promoter-mediated oncogene activation and transcriptional diversity in cancer, which may be exploited therapeutically.

Having the cake and eating it? Clofazimine boosts immunotherapy while limiting side effects.

Combined immune checkpoint blockade (ICB) for cancer exhibits good efficacy in a subset of patients but also associates with immune-related adverse events. Xue et al. use an elegant drug screening strategy to identify the antimicrobial drug clofazimine as an agent that both potentiates ICB efficacy and decreases immune-related adverse events.

Immunologic Profiling of Immune-Related Cutaneous Adverse Events with Checkpoint Inhibitors Reveals Polarized Actionable Pathways.

PURPOSE: Immune-related cutaneous adverse events (ircAE) occur in ≥50% of patients treated with checkpoint inhibitors, but the underlying mechanisms for ircAEs are poorly understood. EXPERIMENTAL DESIGN: Phenotyping/biomarker analyses were conducted in 200 patients on checkpoint inhibitors [139 with ircAEs and 61 without (control group)] to characterize their clinical presentation and immunologic endotypes. Cytokines were evaluated in skin biopsies, skin tape strip extracts, and plasma using real-time PCR and Meso Scale Discovery multiplex cytokine assays. RESULTS: Eight ircAE phenotypes were identified: pruritus (26%), maculopapular rash (MPR; 21%), eczema (19%), lichenoid (11%), urticaria (8%), psoriasiform (6%), vitiligo (5%), and bullous dermatitis (4%). All phenotypes showed skin lymphocyte and eosinophil infiltrates. Skin biopsy PCR revealed the highest increase in IFNγ mRNA in patients with lichenoid (P < 0.0001) and psoriasiform dermatitis (P < 0.01) as compared with patients without ircAEs, whereas the highest IL13 mRNA levels were detected in patients with eczema (P < 0.0001, compared with control). IL17A mRNA was selectively increased in psoriasiform (P < 0.001), lichenoid (P < 0.0001), bullous dermatitis (P < 0.05), and MPR (P < 0.001) compared with control. Distinct cytokine profiles were confirmed in skin tape strip and plasma. Analysis determined increased skin/plasma IL4 cytokine in pruritus, skin IL13 in eczema, plasma IL5 and IL31 in eczema and urticaria, and mixed-cytokine pathways in MPR. Broad inhibition via corticosteroids or type 2 cytokine-targeted inhibition resulted in clinical benefit in these ircAEs. In contrast, significant skin upregulation of type 1/type 17 pathways was found in psoriasiform, lichenoid, bullous dermatitis, and type 1 activation in vitiligo. CONCLUSIONS: Distinct immunologic ircAE endotypes suggest actionable targets for precision medicine-based interventions.

Synthetic dual co-stimulation increases the potency of HIT and TCR-targeted cell therapies.

Chimeric antigen receptor T cells have dramatically improved the treatment of hematologic malignancies. T cell antigen receptor (TCR)-based cell therapies are yet to achieve comparable outcomes. Importantly, chimeric antigen receptors not only target selected antigens but also reprogram T cell functions through the co-stimulatory pathways that they engage upon antigen recognition. We show here that a fusion receptor comprising the CD80 ectodomain and the 4-1BB cytoplasmic domain, termed 80BB, acts as both a ligand and a receptor to engage the CD28 and 4-1BB pathways, thereby increasing the antitumor potency of human leukocyte antigen-independent TCR (HIT) receptor- or TCR-engineered T cells and tumor-infiltrating lymphocytes. Furthermore, 80BB serves as a switch receptor that provides agonistic 4-1BB co-stimulation upon its ligation by the inhibitory CTLA4 molecule. By combining multiple co-stimulatory features in a single antigen-agnostic synthetic receptor, 80BB is a promising tool to sustain CD3-dependent T cell responses in a wide range of targeted immunotherapies.

Preoperative immune checkpoint inhibition and cryoablation in early-stage breast cancer.

Local cryoablation can engender systemic immune activation/anticancer responses in tumors otherwise resistant to immune checkpoint blockade (ICB). We evaluated the safety/tolerability of preoperative cryoablation plus ipilimumab and nivolumab in 5 early-stage/resectable breast cancers. The primary endpoint was met when all 5 patients underwent standard-of-care primary breast surgery undelayedly. Three patients developed transient hyperthyroidism; one developed grade 4 liver toxicity (resolved with supportive management). We compared this strategy with cryoablation and/or ipilimumab. Dual ICB plus cryoablation induced higher expression of T cell activation markers and serum Th1 cytokines and reduced immunosuppressive serum CD4+PD-1hi T cells, improving effector-to-suppressor T cell ratio. After dual ICB and before cryoablation, T cell receptor sequencing of 4 patients showed increased T cell clonality. In this small subset of patients, we provide preliminary evidence that preoperative cryoablation plus ipilimumab and nivolumab is feasible, inducing systemic adaptive immune activation potentially more robust than cryoablation with/without ipilimumab.

Clinical and molecular features of acquired resistance to immunotherapy in non-small cell lung cancer.

Although immunotherapy with PD-(L)1 blockade is routine for lung cancer, little is known about acquired resistance. Among 1,201 patients with non-small cell lung cancer (NSCLC) treated with PD-(L)1 blockade, acquired resistance is common, occurring in >60% of initial responders. Acquired resistance shows differential expression of inflammation and interferon (IFN) signaling. Relapsed tumors can be separated by upregulated or stable expression of IFNγ response genes. Upregulation of IFNγ response genes is associated with putative routes of resistance characterized by signatures of persistent IFN signaling, immune dysfunction, and mutations in antigen presentation genes which can be recapitulated in multiple murine models of acquired resistance to PD-(L)1 blockade after in vitro IFNγ treatment. Acquired resistance to PD-(L)1 blockade in NSCLC is associated with an ongoing, but altered IFN response. The persistently inflamed, rather than excluded or deserted, tumor microenvironment of acquired resistance may inform therapeutic strategies to effectively reprogram and reverse acquired resistance.

Enabling continuous immune cell recirculation on a microfluidic array to study immunotherapeutic interactions in a recapitulated tumour microenvironment.

The effects of immunotherapeutics on interactions between immune and cancer cells are modulated by multiple components in the tumour microenvironment (TME), including endothelium and tumour stroma, which provide both a physical barrier and immunosuppressive stimuli. Herein, we report a recirculating chip to enable continuous immune cell recirculation through a microfluidic cell array to include these crucial players. This system consists of a three-layered cell array (µFCA) spatially emulating the TME, with tailored fluidic circuits establishing T cell recirculation. This platform enables the study of dynamics among the TME, immune cells in a circulatory system and cancer cell responses thereof. Through this system, we found that tumour endothelium hindered T cell infiltration into the reconstructed breast cancer tumour compartment. This negative effect was alleviated when treated with anti-human PD-L1 (programmed cell death ligand 1) antibody. Another key stromal component - cancer associated fibroblasts - attenuated T cell infiltration, compared against normal fibroblasts, and led to reduced apoptotic activity in cancer cells. These results confirm the capability of our tumour-on-a-chip system in identifying some key axes to target in overcoming barriers to immunotherapy by recapitulating immune cell interactions with the reconstructed TME. Our results also attest to the feasibility of scaling up this system for high-throughput cancer immunotherapeutic screening.

APR-246 increases tumor antigenicity independent of p53.

We previously reported that activation of p53 by APR-246 reprograms tumor-associated macrophages to overcome immune checkpoint blockade resistance. Here, we demonstrate that APR-246 and its active moiety, methylene quinuclidinone (MQ) can enhance the immunogenicity of tumor cells directly. MQ treatment of murine B16F10 melanoma cells promoted activation of melanoma-specific CD8+ T cells and increased the efficacy of a tumor cell vaccine using MQ-treated cells even when the B16F10 cells lacked p53. We then designed a novel combination of APR-246 with the TLR-4 agonist, monophosphoryl lipid A, and a CD40 agonist to further enhance these immunogenic effects and demonstrated a significant antitumor response. We propose that the immunogenic effect of MQ can be linked to its thiol-reactive alkylating ability as we observed similar immunogenic effects with the broad-spectrum cysteine-reactive compound, iodoacetamide. Our results thus indicate that combination of APR-246 with immunomodulatory agents may elicit effective antitumor immune response irrespective of the tumor's p53 mutation status.

Phase Ib Trial of Phenformin in Patients with V600-mutated Melanoma Receiving Dabrafenib and Trametinib.

PURPOSE: Preclinical studies show that activation of AMP kinase by phenformin can augment the cytotoxic effect and RAF inhibitors in BRAF V600-mutated melanoma. We conducted a phase Ib dose-escalation trial of phenformin with standard dose dabrafenib/trametinib in patients with metastatic BRAF V600-mutated melanoma. EXPERIMENTAL DESIGN: We used a 3+3 dose-escalation design which explored phenformin doses between 50 and 200 mg twice daily. Patients also received standard dose dabrafenib/trametinib. We measured phenformin pharmacokinetics and assessed the effect of treatment on circulating myeloid-derived suppressor cells (MDSC). RESULTS: A total of 18 patients were treated at dose levels ranging from 50 to 200 mg twice daily. The planned dose-escalation phase had to be cancelled because of the COVID 19 pandemic. The most common toxicities were nausea/vomiting; there were two cases of reversible lactic acidosis. Responses were seen in 10 of 18 patients overall (56%) and in 2 of 8 patients who had received prior therapy with RAF inhibitor. Pharmacokinetic data confirmed drug bioavailability. MDSCs were measured in 7 patients treated at the highest dose levels and showed MDSC levels declined on study drug in 6 of 7 patients. CONCLUSIONS: We identified the recommended phase II dose of phenformin as 50 mg twice daily when administered with dabrafenib/trametinib, although some patients will require short drug holidays. We observed a decrease in MDSCs, as predicted by preclinical studies, and may enhance immune recognition of melanoma cells. SIGNIFICANCE: This is the first trial using phenformin in combination with RAF/MEK inhibition in patients with BRAF V600-mutated melanoma. This is a novel strategy, based on preclinical data, to increase pAMPK while blocking the MAPK pathway in melanoma. Our data provide justification and a recommended dose for a phase II trial.

Determinants of Survival with Combined HER2 and PD-1 Blockade in Metastatic Esophagogastric Cancer.

PURPOSE: We report updated clinical outcomes from a phase II study of pembrolizumab, trastuzumab, and chemotherapy (PTC) in metastatic esophagogastric cancer in conjunction with outcomes from an independent Memorial Sloan Kettering (MSK) cohort. PATIENTS AND METHODS: The significance of pretreatment 89Zr-trastuzumab PET, plasma circulating tumor DNA (ctDNA) dynamics, and tumor HER2 expression and whole exome sequencing was evaluated to identify prognostic biomarkers and mechanisms of resistance in patients treated on-protocol with PTC. Additional prognostic features were evaluated using a multivariable Cox regression model of trastuzumab-treated MSK patients (n = 226). Single-cell RNA sequencing (scRNA-seq) data from MSK and Samsung were evaluated for mechanisms of therapy resistance. RESULTS: 89Zr-trastuzumab PET, scRNA-seq, and serial ctDNA with CT imaging identified how pre-treatment intrapatient genomic heterogeneity contributes to inferior progression-free survival (PFS). We demonstrated that the presence of intensely avid lesions by 89Zr-trastuzumab PET declines in tumor-matched ctDNA by 3 weeks, and clearance of tumor-matched ctDNA by 9 weeks were minimally invasive biomarkers of durable PFS. Paired pre- and on-treatment scRNA-seq identified rapid clearance of HER2-expressing tumor clones with expansion of clones expressing a transcriptional resistance program, which was associated with MT1H, MT1E, MT2A, and MSMB expression. Among trastuzumab-treated patients at MSK, ERBB2 amplification was associated with improved PFS, while alterations in MYC and CDKN2A/B were associated with inferior PFS. CONCLUSIONS: These findings highlight the clinical relevance of identifying baseline intrapatient heterogeneity and serial ctDNA monitoring of HER2-positive esophagogastric cancer patients to identify early evidence of treatment resistance, which could guide proactive therapy escalation or deescalation.

Correction: Multiplexed molecular imaging with surface enhanced resonance Raman scattering nanoprobes reveals immunotherapy response in mice via multichannel image segmentation.

Correction for 'Multiplexed molecular imaging with surface enhanced resonance Raman scattering nanoprobes reveals immunotherapy response in mice via multichannel image segmentation' by Chrysafis Andreou et al., Nanoscale Horiz., 2022, 7, 1540-1552, https://doi.org/10.1039/d2nh00331g.

MCT4-dependent lactate secretion suppresses antitumor immunity in LKB1-deficient lung adenocarcinoma.

Inactivating STK11/LKB1 mutations are genomic drivers of primary resistance to immunotherapy in KRAS-mutated lung adenocarcinoma (LUAD), although the underlying mechanisms remain unelucidated. We find that LKB1 loss results in enhanced lactate production and secretion via the MCT4 transporter. Single-cell RNA profiling of murine models indicates that LKB1-deficient tumors have increased M2 macrophage polarization and hypofunctional T cells, effects that could be recapitulated by the addition of exogenous lactate and abrogated by MCT4 knockdown or therapeutic blockade of the lactate receptor GPR81 expressed on immune cells. Furthermore, MCT4 knockout reverses the resistance to PD-1 blockade induced by LKB1 loss in syngeneic murine models. Finally, tumors from STK11/LKB1 mutant LUAD patients demonstrate a similar phenotype of enhanced M2-macrophages polarization and hypofunctional T cells. These data provide evidence that lactate suppresses antitumor immunity and therapeutic targeting of this pathway is a promising strategy to reversing immunotherapy resistance in STK11/LKB1 mutant LUAD.

Personalized RNA neoantigen vaccines stimulate T cells in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is lethal in 88% of patients1, yet harbours mutation-derived T cell neoantigens that are suitable for vaccines 2,3. Here in a phase I trial of adjuvant autogene cevumeran, an individualized neoantigen vaccine based on uridine mRNA-lipoplex nanoparticles, we synthesized mRNA neoantigen vaccines in real time from surgically resected PDAC tumours. After surgery, we sequentially administered atezolizumab (an anti-PD-L1 immunotherapy), autogene cevumeran (a maximum of 20 neoantigens per patient) and a modified version of a four-drug chemotherapy regimen (mFOLFIRINOX, comprising folinic acid, fluorouracil, irinotecan and oxaliplatin). The end points included vaccine-induced neoantigen-specific T cells by high-threshold assays, 18-month recurrence-free survival and oncologic feasibility. We treated 16 patients with atezolizumab and autogene cevumeran, then 15 patients with mFOLFIRINOX. Autogene cevumeran was administered within 3 days of benchmarked times, was tolerable and induced de novo high-magnitude neoantigen-specific T cells in 8 out of 16 patients, with half targeting more than one vaccine neoantigen. Using a new mathematical strategy to track T cell clones (CloneTrack) and functional assays, we found that vaccine-expanded T cells comprised up to 10% of all blood T cells, re-expanded with a vaccine booster and included long-lived polyfunctional neoantigen-specific effector CD8+ T cells. At 18-month median follow-up, patients with vaccine-expanded T cells (responders) had a longer median recurrence-free survival (not reached) compared with patients without vaccine-expanded T cells (non-responders; 13.4 months, P = 0.003). Differences in the immune fitness of the patients did not confound this correlation, as responders and non-responders mounted equivalent immunity to a concurrent unrelated mRNA vaccine against SARS-CoV-2. Thus, adjuvant atezolizumab, autogene cevumeran and mFOLFIRINOX induces substantial T cell activity that may correlate with delayed PDAC recurrence.

OX40L-expressing recombinant modified vaccinia virus Ankara induces potent antitumor immunity via reprogramming Tregs.

Effective depletion of immune suppressive regulatory T cells (Tregs) in the tumor microenvironment without triggering systemic autoimmunity is an important strategy for cancer immunotherapy. Modified vaccinia virus Ankara (MVA) is a highly attenuated, non-replicative vaccinia virus with a long history of human use. Here, we report rational engineering of an immune-activating recombinant MVA (rMVA, MVA∆E5R-Flt3L-OX40L) with deletion of the vaccinia E5R gene (encoding an inhibitor of the DNA sensor cyclic GMP-AMP synthase, cGAS) and expression of two membrane-anchored transgenes, Flt3L and OX40L. Intratumoral (IT) delivery of rMVA (MVA∆E5R-Flt3L-OX40L) generates potent antitumor immunity, dependent on CD8+ T cells, the cGAS/STING-mediated cytosolic DNA-sensing pathway, and type I IFN signaling. Remarkably, IT rMVA (MVA∆E5R-Flt3L-OX40L) depletes OX40hi regulatory T cells via OX40L/OX40 interaction and IFNAR signaling. Single-cell RNA-seq analyses of tumors treated with rMVA showed the depletion of OX40hiCCR8hi Tregs and expansion of IFN-responsive Tregs. Taken together, our study provides a proof-of-concept for depleting and reprogramming intratumoral Tregs via an immune-activating rMVA.

T cell immunotherapies engage neutrophils to eliminate tumor antigen escape variants.

Cancer immunotherapies, including adoptive T cell transfer, can be ineffective because tumors evolve to display antigen-loss-variant clones. Therapies that activate multiple branches of the immune system may eliminate escape variants. Here, we show that melanoma-specific CD4+ T cell therapy in combination with OX40 co-stimulation or CTLA-4 blockade can eradicate melanomas containing antigen escape variants. As expected, early on-target recognition of melanoma antigens by tumor-specific CD4+ T cells was required. Surprisingly, complete tumor eradication was dependent on neutrophils and partly dependent on inducible nitric oxide synthase. In support of these findings, extensive neutrophil activation was observed in mouse tumors and in biopsies of melanoma patients treated with immune checkpoint blockade. Transcriptomic and flow cytometry analyses revealed a distinct anti-tumorigenic neutrophil subset present in treated mice. Our findings uncover an interplay between T cells mediating the initial anti-tumor immune response and neutrophils mediating the destruction of tumor antigen loss variants.

Lineage tracing reveals clonal progenitors and long-term persistence of tumor-specific T cells during immune checkpoint blockade.

Paired single-cell RNA and T cell receptor sequencing (scRNA/TCR-seq) has allowed for enhanced resolution of clonal T cell dynamics in cancer. Here, we report a scRNA/TCR-seq analysis of 187,650 T cells from 31 tissue regions, including tumor, adjacent normal tissues, and lymph nodes (LN), from three patients with non-small cell lung cancer after immune checkpoint blockade (ICB). Regions with viable cancer cells are enriched for exhausted CD8+ T cells, regulatory CD4+ T cells (Treg), and follicular helper CD4+ T cells (TFH). Tracking T cell clonotypes across tissues, combined with neoantigen specificity assays, reveals that TFH and tumor-specific exhausted CD8+ T cells are clonally linked to TCF7+SELL+ progenitors in tumor draining LNs, and progressive exhaustion trajectories of CD8+ T, Treg, and TFH cells with proximity to the tumor microenvironment. Finally, longitudinal tracking of tumor-specific CD8+ and CD4+ T cell clones reveals persistence in the peripheral blood for years after ICB therapy.