Combined Blockade Research Articles

Dual versus single immune checkpoint blockade for patients with metastatic colorectal cancer harboring microsatellite instability: The Saint-Antoine immunoMSI score.

168 Background: Anti-PD1 monotherapy (mono) and anti-PD1 plus anti-CTLA4 therapies (combo) are validated for metastatic colorectal cancer (mCRC) harboring microsatellite instability and/or mismatch repair deficiency (MSI/dMMR). We aimed at assessing the efficacy of combined anti-CTLA4 and anti-PD1 blockade versus anti-PD1 monotherapy and identifying which subgroups benefit more from the combo. Methods: All patients (pts) with MSI/dMMR mCRC treated by anti-PD1 ± anti-CTLA4 included in the prospective monocenter immunoMSI cohort were analyzed. The choice between monotherapy or combination therapy varied according to open studies and regulatory approvals, without taking pt characteristics into account. Datalock date was April 23, 2024. Main endpoints were progression-free survival (PFS) per iRECIST and overall survival (OS) estimated with the Kaplan-Meier method. Univariate Cox proportional hazards models were used to estimate hazard ratios with CI. The interactions between studied subgroups and the treatment group were modelized by adding in a single Cox model the studied subgroups, the treatment group parameters, and their interaction term. Results: Among 210 pts, 97 were treated combo and 113 with mono. Baseline characteristics were similar in both groups. 25% and 17% were in first line, respectively. Median follow-up was 4.4 years: 4.1 years for combo and 5.4 years for mono. Combo was associated with better PFS (HR=0.48; 95%CI 0.31-0.76) and OS (HR=0.49; 0.29-0.84). Best observed response rates were: 78% of objective responses, 17% of stable diseases and 5% disease progressions with combo versus 60%, 25% and 15% with mono. Subgroup analyses showed significant differential effect of the treatment group for gender (PFS HR=0.21 vs 0.87 for females and males, P-interaction: 0.0067), liver metastases (PFS HR=0.30 vs 0.79 for pts without liver mets and those with liver mets, P-interaction: 0.0479) and sidedness (PFS HR=0.27 vs 0.64 for left-sided and right-sided mCRC, P-interaction=0.0922), but not RAS / RAF mutations. We computed a predictive score based on gender (female: 2 points), no liver met (1 point) and left side (1 point). PFS HR was 0.1 (0.03-0.33) for the group ≥3 points (N=67), 0.66 (0.36-1.23) for the group with 1-2 points (N=113) and 1.54 (0.50-4.70) for the group with 0 point (N=30). Conclusions: Combined CTLA4 and PD1 blockade is associated with improved PFS and OS compared to anti-PD1 alone in MSI/dMMR mCRC. The subgroup deriving the highest benefit of the combo seems to be female pts with left sided tumor and no liver met. Data from the randomization between nivolumab versus nivolumab + ipilumumab in the CheckMate-8HW phase III study are expected.

YAP1 induces bladder cancer progression and promotes immune evasion through IL-6/STAT3 pathway and CXCL deregulation.

The Hippo signaling pathway plays a key role in tumorigenesis in different cancer types. We investigated the role of the Hippo effector YAP1 in the tumor immune microenvironment (TIME) of urothelial carcinoma of the bladder (UCB) and evaluated the efficacy of immunotherapy in the context of YAP1 signaling. We performed numerous in vitro and in vivo experiments to determine the role of YAP1 using genetic and pharmacological attenuation of YAP1 activity. Briefly, RNA sequencing was carried out with mouse and human cell lines to identify novel YAP1-regulated downstream targets unbiasedly. We then experimentally confirmed that YAP1 regulates the TIME through the IL-6/STAT3 signaling pathway and varied C-X-C motif chemokine regulation. We analyzed several human sample sets to explore the TIME status in the context of YAP1 expression. Our data indicate that YAP1 attenuation decreases M2 macrophages and myeloid-derived suppressor cells in the TIME compared with YAP1-expressing cells. In summary, this study provides insights into YAP1 signaling as a driver for cancer stemness and an inducer of immunosuppressive TIME. Moreover, the therapeutic efficacy of YAP1 attenuation indicates that combined blockade of YAP1 and immune checkpoints may yield clinical value for treating patients with UCB.

Combined PD-1 and IL-10 blockade reinvigorates mucosal CD8+T exhaustion and relieves liver damage after intestinal ischemia reperfusion attack

Currently, impairments in gut mucosal immunity following intestinal ischemia reperfusion (IR) remain unclear. Mucosal CD8+T cells are critical for host defense against bacterial translocation from the gut lumen, and exhausted T cells lose robust effector functions. The present study was designed to verify the hypothesis that intestinal IR leads to mucosal CD8+T cell exhaustion, and that reinvigoration of exhausted CD8+T cell attenuates IR-induced bacterial translocation and liver damage. The intestinal IR model was performed through clamping the superior mesenteric artery in mice. The percent of exhausted CD8+T cells and the effector function of CD8+T cells were examined to determine the occurrence of intestinal mucosal CD8+T cell exhaustion. Subsequently, PD-1 blockade or combined PD-1 and IL-10 blockade was respectively used to reinvigorate exhausted CD8+T cells. Serum biomarkers, bacterial RNA and colonies, and inflammatory factors were examined to determine bacterial translocation and liver damage. The results indicated that intestinal IR induced CD8+T cell exhaustion in mucosal tissues, as evidenced by increased PD-1+ and PD-1+LAG-3+CD8+T cells and decreased IL-2 and TNF-α expression in CD8+T cells. Combined PD-1 and IL-10 blockade, but not PD-1 blockade alone, reinvigorated CD8+T cell exhaustion, as evidenced by increased generation of exhausted CD8+T cells with cytotoxicity and effector function, and elevated production of IFN-γ. Moreover, combined blockade significantly reduced the translocation of gut bacteria and injury to the liver after IR. In conclusion, intestinal IR leads to mucosal CD8+T cell exhaustion. Combined PD-1 and IL-10 blockade reinvigorates exhausted CD8+T cells, and ameliorates bacterial translocation and liver damage following IR.

Combined PARP14 inhibition and PD-1 blockade promotes cytotoxic T cell quiescence and modulates macrophage polarization in relapsed melanoma

BackgroundProgrammed cell death 1 (PD-1) signaling blockade by immune checkpoint inhibitors (ICI) effectively restores immune surveillance to treat melanoma. However, chronic interferon-gamma (IFNγ)-induced immune homeostatic responses in melanoma cells contribute...

Circulating tumor cell plasticity determines breast cancer therapy resistance via neuregulin 1–HER3 signaling

Circulating tumor cells (CTCs) drive metastasis, the leading cause of death in individuals with breast cancer. Due to their low abundance in the circulation, robust CTC expansion protocols are urgently needed to effectively study disease progression and therapy responses. Here we present the establishment of long-term CTC-derived organoids from female individuals with metastatic breast cancer. Multiomics analysis of CTC-derived organoids along with preclinical modeling with xenografts identified neuregulin 1 (NRG1)–ERBB2 receptor tyrosine kinase 3 (ERBB3/HER3) signaling as a key pathway required for CTC survival, growth and dissemination. Genome-wide CRISPR activation screens revealed that fibroblast growth factor receptor 1 (FGFR1) signaling serves a compensatory function to the NRG1–HER3 axis and rescues NRG1 deficiency in CTCs. Conversely, NRG1–HER3 activation induced resistance to FGFR1 inhibition, whereas combinatorial blockade impaired CTC growth. The dynamic interplay between NRG1–HER3 and FGFR1 signaling reveals the molecular basis of cancer cell plasticity and clinically relevant strategies to target it. Our CTC organoid platform enables the identification and validation of patient-specific vulnerabilities and represents an innovative tool for precision medicine.

Separate and combined blockades of α- and β-adrenergic receptors in forearm sweating induced by adrenergic agents and exercise in the heat in young adults.

The assessment of adrenergic modulation of sweating as assessed via pharmacologic administration of α- and β-adrenergic receptor blockers during exercise has yielded mixed findings. However, the underlying mechanisms for this disparity remains unresolved. We investigated the effects of separate and combined blockade of α- and β-adrenergic receptors on forearm sweating induced by a 30-min moderate-intensity exercise bout (n=17, protocol 1) and the administration of adrenergic agonists epinephrine and norepinephrine (n=16, protocol 2) in the heat. Adrenergic receptor blockade was induced via the separate and combined iontophoretic administration of terazosin (α-adrenergic receptor antagonist) and propranolol (β-adrenergic receptor antagonist) on forearm skin. Bretylium, a noradrenergic sympathetic nerve inhibitor was also administered separately in protocol 1. In protocol 1, relative to the separate administration of propranolol, terazosin alone or in combination with propranolol attenuated exercise sweating to a similar extent (both P£0.037), although the effect was reduced relative to that observed with bretylium treatment (P<0.001). In protocol 2, administration of propranolol increased norepinephrine- (P=0.029) but not epinephrine-induced sweat rate. The combined administration of terazosin reversed this response, attenuating sweating (P<0.001) to a greater extent than terazosin treatment alone (P=0.030). Altogether, we showed that while β-adrenergic receptors may interact with α-adrenergic receptors pharmacologically, it does not appear to modulate exercise-induced sweating on the forearm. Further, α- but not β-adrenergic receptors independently modulate the regulation of forearm sweating during exercise in the heat. Finally, the bretylium-induced reduction in forearm sweat rate during exercise likely occurs independently of α- and β-adrenergic receptors.

Understanding the PULSAR effect in combined radiotherapy and immunotherapy using transformer-based attention mechanisms.

PULSAR (personalized, ultra-fractionated stereotactic adaptive radiotherapy) is the adaptation of stereotactic ablative radiotherapy towards personalized cancer management. It has potential to harness the synergy between radiation therapy and immunotherapy, such as immune checkpoint inhibitors to amplify the anti-tumor immune response. For the first time, we applied a transformer-based attention mechanism to investigate the underlying interactions between combined PULSAR and PD-L1 blockade immunotherapy, based on the preliminary experimental results of a murine cancer model (Lewis Lung Carcinoma, LLC). The radiation and administration of α-PD-L1 were viewed as two external stimulation signals occurring in a temporal sequence. Our study demonstrates the utility of a transformer model in 1) predicting tumor changes in response to specific treatment schemes, and 2) generating self-attention and cross-attention maps. The cross-attention maps serve as a biological representation of the semantic similarity between source and target sentences in neural translation, offering insights into the causal relationships of the PULSAR effect. Our model offers a unique perspective with the potential to enhance the understanding of the temporal dependencies of the PULSAR effect on time, dose, and T cell dynamics. In a broader context, our proposed framework offers the potential to explore varying intervals and doses for subsequent treatments while monitoring the biological parameters impacted by these perturbations. This approach can lead to more personalized and rational radiation or drug interactions.

TIGIT and PD-L1 co-blockade promotes clonal expansion of multipotent, non-exhausted antitumor T cells by facilitating co-stimulation

Blockade of immune checkpoints PD-1 and TIGIT has demonstrated activity in mouse tumor models and human patients with cancer. Although these coinhibitory receptors can restrict signaling in CD8+ T cells by regulating their associated co-stimulatory receptors CD28 and CD226, the functional consequences of combining PD-1 and TIGIT blockade remain poorly characterized. In mouse tumor models, we show that combination blockade elicited CD226-driven clonal expansion of tumor antigen-specific CD8+ T cells. The expanded clones emerged from a population of stem-like cells in draining lymph nodes, entering the blood as a previously unidentified single-phenotype, multiclonal population. Upon reaching the tumor, these transiting cells expanded further and differentiated into effector or exhausted T cells, with combination blockade restricting entry into the exhaustion pathway by favoring co-stimulation. Thus, PD-1 and TIGIT inhibition helps shape the repertoire of tumor-reactive CD8+ T cells in draining lymph nodes and determines their immunological fate in the tumor to enhance therapeutic benefit. Analysis of clinical trial samples suggests a similar mechanism may also occur in patients with cancer.

USP1 deubiquitinates PARP1 to regulate its trapping and PARylation activity.

PARP inhibitors (PARPi) represent a game-changing treatment for patients with ovarian cancer with tumors deficient for the homologous recombination (HR) pathway treated with platinum (Pt)-based therapy. PARPi exert their cytotoxic effect by both trapping PARP1 on the damaged DNA and by restraining its enzymatic activity (PARylation). How PARP1 is recruited and trapped at the DNA damage sites and how resistance to PARPi could be overcome are still matters of investigation. Here, we described PARP1 as a substrate of the deubiquitinase USP1. At molecular level, USP1 binds PARP1 to remove its K63-linked polyubiquitination and controls PARP1 chromatin trapping and PARylation activity, regulating sensitivity to PARPi. In both Pt/PARPi-sensitive and -resistant cells, USP1/PARP1 combined blockade enhances replicative stress, DNA damage, and cell death. Our work dissected the biological interaction between USP1 and PARP1 and recommended this axis as a promising and powerful therapeutic choice for not only sensitive but also chemoresistant patients with ovarian cancer irrespective of their HR status.

Combination CXCR4 and PD1 blockade enhances intratumoral dendritic cell activation and immune responses against hepatocellular carcinoma.

Immune checkpoint inhibitors (ICIs) have revolutionized the treatment of unresectable hepatocellular carcinoma (HCC), but their impressive efficacy is seen in just a fraction of patients. One key mechanism of immunotherapy resistance is the paucity of dendritic cells (DCs) in liver malignancies. Here, we tested combination blockade of programmed death receptor 1 (PD1) and CXCR4, a receptor for CXCL12, a pleiotropic factor that mediates immunosuppression in tumors. Using orthotopic grafted and autochthonous HCC models with underlying liver damage, we evaluated treatment feasibility and efficacy. In addition, we examined the effects of treatment using immunofluorescence, flow cytometric analysis of DCs in vivo and in vitro, and RNA-sequencing. Combination anti-CXCR4 and anti-PD1therapy was safe and significantly inhibited tumor growth and prolonged survival in all murine preclinical models of HCC tested. The combination treatment successfully reprogrammed antigen-presenting cells, revealing the potential role of conventional type 1 DCs (cDC1s) in the HCC microenvironment. Moreover, DC reprogramming enhanced anticancer immunity by facilitating CD8+ T-cell accumulation and activation in the HCC tissue. The effectiveness of anti-CXCR4/PD1 therapy was compromised entirely in Batf3-KO mice deficient in cDC1s. Thus, combined CXCR4/PD1 blockade can reprogram intra-tumoral cDC1s and holds the potential to potentiate antitumor immune response against HCC.

Natural killer cells are required for the recruitment of CD8+ T cells and the efficacy of immune checkpoint blockade in melanoma brain metastases

Background Brain metastases (BrM) affect up to 60% of patients with metastatic melanoma and are associated with poor prognosis. While combined immune checkpoint blockade of programmed death-1 (PD-1) and cytotoxic...

Impact of Bone Metastasis in Stage IV Non–Small Cell Lung Cancer Treated With Durvalumab and Tremelimumab With or Without Chemotherapy: A Retrospective Analysis of the CCTG BR.34 Trial

PURPOSE This retrospective analysis examines the impact of bone metastasis on outcomes in patients with non–small cell lung cancer (NSCLC) from the CCTG BR.34 trial, which investigated the combined immune checkpoint blockade. MATERIALS AND METHODS The CCTG BR.34 trial was a randomized phase II study assessing durvalumab plus tremelimumab, with or without platinum-doublet chemotherapy, in 301 patients with metastatic NSCLC. Patients were categorized into two cohorts on the basis of bone metastasis: cohort A (n = 129) and cohort B (n = 172). The primary end point was overall survival (OS), analyzed using Cox regression and multivariable models. RESULTS Patients with bone metastasis had notably poorer outcomes. The median OS was 10.9 months for those with bone metastasis versus 18.7 months for those without (hazard ratio [HR] 1.68, P = .001). The median progression-free survival (PFS) was 3.4 months with bone metastasis compared with 7.2 months without (HR, 1.82, P &lt; .0001). The overall response rate (ORR) was lower in patients with bone metastasis (29.5%) compared with those without (45.9%; odds ratio [OR], 0.52, P = .01). Adding chemotherapy to durvalumab plus tremelimumab did not significantly affect OS ( P = .23), PFS ( P = .84), or ORR ( P = .25) in relation to bone metastasis. Multivariable analysis reaffirmed that bone metastasis was linked to decreased OS (HR, 1.42, P = .036), PFS (HR, 1.69, P &lt; .0001), and ORR (OR, 0.52, P = .01). CONCLUSION Bone metastasis was associated with worse outcomes in this dual immune checkpoint blockade trial, with or without chemotherapy. Future trials should consider bone metastasis as a stratification factor and explore combining immune checkpoint inhibitors with targeted therapies addressing bone microenvironment factors (eg, interleukin-8, PTHrP, and transforming growth factor-β).

Combined programmed cell death protein 1 and cytotoxic T-lymphocyte associated protein 4 blockade in an international cohort of patients with acral lentiginous melanoma.

Combination immune checkpoint blockade targeting programmed cell death protein 1 (PD-1) and cytotoxic T-lymphocyte associated protein 4 (CTLA-4) leads to high response rates and improved survival in patients with advanced cutaneous melanoma (CM). Less is known about the efficacy of this combination in acral lentiginous melanoma (ALM). To determine the efficacy of combination immune checkpoint blockade targeting PD-1 and CTLA-4 in a diverse, real-world population of patients with ALM. This multi-institutional retrospective study analysed patients with histologically confirmed ALM treated with a combination of PD-1 and CTLA-4 inhibitors between 2010 and 2022. The primary objective of the study was the objective response rate (ORR) as per the RECIST criteria. The secondary objectives were progression-free survival (PFS) and overall survival (OS). In total, 109 patients with advanced ALM treated with combined PD-1 and CTLA-4 blockade in any line of treatment were included. The majority of patients had stage IV disease (n = 81; 74.3%). The ORR for the entire cohort was 18.3% [95% confidence interval (CI) 11.6-26.9], with 9 (8.3%) complete and 11 (10.1%) partial responses. A further 22 patients (20.2%) had stable disease, and the disease control rate was 38.5%. Median PFS was 4.2 months (95% CI 3.25-5.62), while median OS was 17 months (95% CI 12.4-23.1). Ninety-five patients (87.2%) had a treatment-related adverse event, with 40.4% (n = 44/109) experiencing at least one grade 3 or 4 toxicity. Elevated lactate dehydrogenase (P = 0.04), ≥ 2 lines of prior treatment (P = 0.03) and Asian ethnicity (P = 0.04) were associated with worse OS, while Hispanic/Latino ethnicity was associated with better OS (P = 0.02). Combination PD-1 and CTLA-4 blockade is less effective for ALM than for CM, despite similar toxicity. In particular, Asian patients appear to derive less benefit from this regimen. Novel treatment approaches are needed for this rare melanoma subtype.

ATR inhibition activates cancer cell cGAS/STING-interferon signaling and promotes antitumor immunity in small-cell lung cancer.

Patients with small-cell lung cancer (SCLC) have poor prognosis and typically experience only transient benefits from combined immune checkpoint blockade (ICB) and chemotherapy. Here, we show that inhibition of ataxia telangiectasia and rad3 related (ATR), the primary replication stress response activator, induces DNA damage-mediated micronuclei formation in SCLC models. ATR inhibition in SCLC activates the stimulator of interferon genes (STING)-mediated interferon signaling, recruits T cells, and augments the antitumor immune response of programmed death-ligand 1 (PD-L1) blockade in mouse models. We demonstrate that combined ATR and PD-L1 inhibition causes improved antitumor response than PD-L1 alone as the second-line treatment in SCLC. This study shows that targeting ATR up-regulates major histocompatibility class I expression in preclinical models and SCLC clinical samples collected from a first-in-class clinical trial of ATR inhibitor, berzosertib, with topotecan in patients with relapsed SCLC. Targeting ATR represents a transformative vulnerability of SCLC and is a complementary strategy to induce STING-interferon signaling-mediated immunogenicity in SCLC.

A covalent creatine kinase inhibitor ablates glioblastoma migration and sensitizes tumors to oxidative stress

Glioblastoma is a Grade 4 primary brain tumor defined by therapy resistance, diffuse infiltration, and near-uniform lethality. The underlying mechanisms are unknown, and no treatment has been curative. Using a recently developed creatine kinase inhibitor (CKi), we explored the role of this inhibitor on GBM biology in vitro. While CKi minimally impacted GBM cell proliferation and viability, it significantly affected migration. In established GBM cell lines and patient-derived xenografts, CKi ablated both the migration and invasion of GBM cells. CKi also hindered radiation-induced migration. RNA-seq revealed a decrease in invasion-related genes, with an unexpected increase in glutathione metabolism and ferroptosis protection genes post-CKi treatment. The effects of CKi could be reversed by the addition of cell-permeable glutathione. Carbon-13 metabolite tracing indicated heightened glutathione biosynthesis post-CKi treatment. Combinatorial CKi blockade and glutathione inhibition or ferroptosis activation abrogated cell survival. Our data demonstrated that CKi perturbs promigratory and anti-ferroptotic roles in GBM, identifying the creatine kinase axis as a druggable target for GBM treatment.

Spatial analysis reveals targetable macrophage-mediated mechanisms of immune evasion in hepatocellular carcinoma minimal residual disease.

Hepatocellular carcinoma (HCC) frequently recurs from minimal residual disease (MRD), which persists after therapy. Here, we identified mechanisms of persistence of residual tumor cells using post-chemoembolization human HCC (n = 108 patients, 1.07 million cells) and a transgenic mouse model of MRD. Through single-cell high-plex cytometric imaging, we identified a spatial neighborhood within which PD-L1 + M2-like macrophages interact with stem-like tumor cells, correlating with CD8+ T cell exhaustion and poor survival. Further, through spatial transcriptomics of residual HCC, we showed that macrophage-derived TGFβ1 mediates the persistence of stem-like tumor cells. Last, we demonstrate that combined blockade of Pdl1 and Tgfβ excluded immunosuppressive macrophages, recruited activated CD8+ T cells and eliminated residual stem-like tumor cells in two mouse models: a transgenic model of MRD and a syngeneic orthotopic model of doxorubicin-resistant HCC. Thus, our spatial analyses reveal that PD-L1+ macrophages sustain MRD by activating the TGFβ pathway in stem-like cancer cells and targeting this interaction may prevent HCC recurrence from MRD.

Influence of RNA Methylation on Cancerous Cells: A Prospective Approach for Alteration of In Vivo Cellular Composition.

RNA methylation is a dynamic and ubiquitous post-transcriptional modification that plays a pivotal role in regulating gene expression in various conditions like cancer, neurological disorders, cardiovascular diseases, viral infections, metabolic disorders, and autoimmune diseases. RNA methylation manifests across diverse RNA species including messenger RNA (mRNA), ribosomal RNA (rRNA), and transfer RNA (tRNA), exerting pivotal roles in gene expression regulation and various biological phenomena. Aberrant activity of writer, eraser, and reader proteins enables dysregulated methylation landscape across diverse malignancy transcriptomes, frequently promoting cancer pathogenesis. Numerous oncogenic drivers, tumour suppressors, invasion/metastasis factors, and signalling cascade components undergo methylation changes that modulate respective mRNA stability, translation, splicing, transport, and protein-RNA interactions accordingly. Functional studies confirm methylation-dependent alterations drive proliferation, survival, motility, angiogenesis, stemness, metabolism, and therapeutic evasion programs systemically. Methyltransferase overexpression typifies certain breast, liver, gastric, and other carcinomas correlating with adverse clinical outcomes like diminished overall survival. Mapping efforts uncover nodal transcripts for targeted drug development against hyperactivated regulators including METTL3. Some erasers and readers also suitable lead candidates based on apparent synthetic lethality. Proteomic screens additionally highlight relevant methylation-sensitive effector pathways amenable to combinatorial blockade, reversing compensatory signalling mechanisms that facilitate solid tumour progression. Quantifying global methylation burdens and responsible enzymes clinically predicts patient prognosis, risk stratification for adjuvant therapy, and overall therapeutic responsiveness.

Combined inhibition of CDK4/6 and AKT is highly effective against the luminal androgen receptor (LAR) subtype of triple negative breast cancer

Luminal Androgen Receptor (LAR) triple-negative breast cancers (TNBC) express androgen receptors (AR), exhibit high frequency of PIK3CA mutations and intact RB. Herein, we investigated combined blockade of the CDK4/6 and PI3K signaling with palbociclib, alpelisib, and capivasertib, which inhibit CDK4/6, PI3Kα, and AKT1-3, respectively. The combination of palbociclib/capivasertib, but not palbociclib/alpelisib, synergistically inhibited proliferation of MDA-MB-453 and MFM-223 LAR cells [synergy score 7.34 (p = 5.81x10−11) and 4.78 (p = 0.012), respectively]. The AR antagonist enzalutamide was inactive against MDA-MB-453, MFM-223, and CAL148 cells and did not enhance the efficacy of either combination. Palbociclib/capivasertib inhibited growth of LAR patient-derived xenografts more potently than palbociclib/alpelisib. Treatment of LAR cells with palbociclib suppressed phosphorylated-RB and resulted in adaptive phosphorylation/activation of S473 pAKT and AKT substrates GSK3β, PRAS40, and FoxO3a. Capivasertib blocked palbociclib-induced phosphorylation of AKT substrates more potently than alpelisib. Treatment with PI3Kβ inhibitors did not block phosphorylation of AKT substrates, suggesting that PI3Kβ did not mediate the adaptive response to CDK4/6 inhibition. Phosphokinase arrays of MDA-MB-453 cells treated with palbociclib showed time-dependent upregulation of PDGFRβ, GSK3β, STAT3, and STAT6. RNA silencing of PDGFRβ in palbociclib-treated MDA-MB-453 and MFM-223 cells blocked the upregulation of S473 pAKT, suggesting that the adaptive response to CDK4/6 blockade involves PDGFRβ signaling. Finally, treatment with palbociclib and the PDGFR inhibitor CP637451 arrested growth of MDA-MB-453 and MFM-223 cells to the same degree as palbociclib/capivasertib. These findings support testing the combination of CDK4/6 and AKT inhibitors in patients with LAR TNBC, and further investigation of PDGFR antagonists in this breast cancer subtype.

Multiplexed Spatial Imaging at the Single-Cell Level Reveals Mutually Exclusive Expression of B7 Family Proteins

Targeting novel inhibitory ligands beyond anti-PD-1 and PD-L1 and CTLA-4 therapies is essential for the next decade of the immunotherapy era. Agents for the B7 family molecules B7-H3, B7-H4, and B7-H5 are emerging in clinical trial phases; therefore, further accumulation of evidence from both clinical and basic aspects is vital. Here, we applied a 7-color multiplexed imaging technique to analyze the profile of B7 family B7-H3/B7-H4/B7-H5 expression, in addition to PD-L1, and the spatial characteristics of immune cell infiltrates in urothelial carcinoma (UC). The results revealed that B7-H3 and B7-H4 were mainly expressed on tumor cells and B7-H5 on immune cells in UC, and most of the B7-H3/B7-H4/B7-H5-positive cells were mutually exclusive with PD-L1-positive cells. Also, the expression of B7-H4 was elevated in patients with advanced pathologic stages, and high B7-H4 expression was a significant factor affecting overall mortality following surgery in UC. Furthermore, spatial analysis revealed that the distance from the B7-H4+ cells to the nearest CD8+ cells was markedly far compared with other B7 family-positive tumor cells. Interestingly, the distance from B7-H4+ cells to the nearest CD8+ cells was significantly farther in patients dying from cancer after surgery or immune checkpoint inhibitors compared with cancer survivors; thus, high B7-H4 expression in tumor cells may inhibit CD8 infiltration into the tumor space and that B7-H4-positive cells form a specific spatial niche. In summary, we performed a comprehensive evaluation of B7 family member expression and found that the spatial distribution of B7-H4 suggests the potentially useful role of combination blockade with both B7-H4 and the current anti-PD-1/PD-L1 axis in the treatment of UC.

HLA-E and NKG2A Mediate Resistance to M. bovis BCG Immunotherapy in Non-Muscle-Invasive Bladder Cancer.

Mycobacterium bovis Bacillus Calmette-Guerin (BCG) is the primary treatment for non-muscle-invasive bladder cancer (NMIBC), known to stimulate inflammatory cytokines, notably interferon (IFN)-γ. We observed that prolonged IFN-γ exposure fosters adaptive resistance in recurrent tumors, aiding immune evasion and tumor proliferation. We identify HLA-E and NKG2A, part of a novel NK and T cell checkpoint pathway, as key mediators of resistance in BCG-unresponsive NMIBC. IFN-γ enhances HLA-E and PD-L1 expression in recurrent tumors, with an enrichment of intra-tumoral NKG2A-expressing NK and CD8 T cells. CXCL9+ macrophages and dendritic cells and CXCL12-expressing stromal cells likely recruit CXCR3/CXCR4-expressing NK and T cells and CXCR7+ HLA-EHIGH tumor cells. NK and CD8 T cells remain functional within BCG-unresponsive tumors but are inhibited by HLA-E and PD-L1, providing a framework for combined NKG2A and PD-L1 blockade strategy for bladder-sparing treatment of BCG-unresponsive NMIBC.