Enhanced anti‐tumor effects of the PD‐1 blockade combined with a highly absorptive form of curcumin targeting STAT3

PD‐1/PD‐L1 immune checkpoint inhibitors are promising cancer immunotherapies however responses are still limited and the development of more effective combination immunotherapy is needed. We previously reported that STAT3 activation in cancer cells and immune cells was involved in immune‐resistant mechanisms. In this study, we evaluated the effect of highly absorptive forms of curcumin extracts and synthetic curcumin on anti‐tumor T cell responses. The curcumin po administration resulted in the significant augmentation of in vivo induction of tumor antigen‐specific T cells through restoration of dendritic cells (DCs) by inhibiting directly STAT3 in DCs and indirectly via reduced IL‐6 production from STAT3 activated cancer cells in 2 syngeneic MC38 and CT26 murine tumor models. Curcumin also showed direct DC enhancing activity and enhanced T cell induction for the immunized antigens in non‐tumor‐bearing mice and human hosts. Curcumin restored DC functions in xenogeneic nude mouse model implanted with high IL‐6‐producing human clear cell ovarian cancer cells. The combination of curcumin and PD‐1/PD‐L1 Abs demonstrated a synergistic anti‐tumor activity in MC38 murine tumor models. These results indicated that curcumin augments the induction of tumor antigen‐specific T cells by restoring the T cell stimulatory activity of DCs targeting activated STAT3 in both cancer cells and immune cells. Combination immunotherapy with curcumin and PD‐1/PD‐L1 Ab is an attractive strategy in the development of effective immunotherapy against various cancers.     

CPI‐203 improves the efficacy of anti‐PD‐1 therapy by inhibiting the induced PD‐L1 overexpression in liver cancer

Hepatocellular carcinoma (HCC) is one of the commonest lethal malignancies worldwide, and often diagnosed at an advanced stage, without any curative therapy. Immune checkpoint blockers targeting the programmed death receptor 1 (PD‐1) have shown impressive antitumor activity in patients with advanced‐stage HCC, while the response rate is only 30%. Inducible PD‐L1 overexpression may result in a lack of response to cancer immunotherapy, which is attributed to a mechanism of adaptive immune resistance. Our study investigated that the overexpression of PD‐L1 promoted the invasion and migration of liver cancer cells in vitro, and the induced overexpression of PD‐L1 in the tumor microenvironment could weaken the effects of anti‐PD‐1 immunotherapy in a BALB/c mouse model of liver cancer. CPI‐203, a small‐molecule bromodomain‐containing protein 4 (BRD4) inhibitor, which can potently inhibit PD‐L1 expression in vitro and in vivo, combined with PD‐1 antibody improved the response to immunotherapy in a liver cancer model. Cell transfection and chromatin immunoprecipitation assay manifested that BRD4 plays a key role in PD‐L1 expression; CPI‐203 can inhibit PD‐L1 expression by inhibiting the BRD4 occupation of the PD‐L1 promoter region. This study indicates a potential clinical immunotherapy method to reduce the incidence of clinical resistance to immunotherapy in patients with HCC.     

Paving the road to make chimeric antigen receptor‐T‐cell therapy effective against solid tumors

The three major standard therapies, that is, surgery, chemotherapy, and radiation therapy have conventionally been applied to the treatments for cancers and have saved many patients. In addition, for intractable, refractory, or advanced malignancies that cannot be cured by the three standard therapies, immunotherapy is an important subject of basic and clinical researches. Immune checkpoint inhibitor therapy (ICI) has shown significant therapeutic efficacies on some types of tumors in large‐scale randomized clinical trials, making a major impact on clinical oncology by scientifically proving and establishing the effectiveness of an immunotherapy. In 2018, ICI was awarded the Nobel Prize in Physiology or Medicine, and immunotherapy is now becoming the “fourth” standard therapy for cancers. Recently, adoptive cell therapies, in which genetically modified T cells with enhanced reactivity against tumors are infused into the patients, have been attracting considerable attention as a hopeful immunotherapy following ICI. Particularly, chimeric antigen receptor (CAR)‐T‐cell therapies demonstrate marked therapeutic efficacies against some hematologic malignancies, and have been approved in many countries. However, current CAR‐T‐cell therapy is considered to be little effective against solid tumors, which is one of the challenging issues to be overcome in CAR‐T‐cell therapy. In this review, we at first introduce CAR and CAR‐T cell, and then focus on the recent progress of CAR‐T‐cell therapy against solid tumors as well as the novel concept on a role of CAR‐T cells, aiming to further understandings of the novel cancer immunotherapies.     

Drug resistance to targeted therapies: Déjà vu all over again

A major limitation of targeted anticancer therapies is intrinsic or acquired resistance. This review emphasizes similarities in the mechanisms of resistance to endocrine therapies in breast cancer and those seen with the new generation of targeted cancer therapeutics. Resistance to single‐agent cancer therapeutics is frequently the result of reactivation of the signaling pathway, indicating that a major limitation of targeted agents lies in their inability to fully block the cancer‐relevant signaling pathway. The development of mechanism‐based combinations of targeted therapies together with non‐invasive molecular disease monitoring is a logical way forward to delay and ultimately overcome drug resistance development.     

Impact of chemotherapy and/or immunotherapy on neutralizing antibody response to SARS‐CoV‐2 mRNA‐1237 vaccine in patients with solid tumors

Patients with solid tumors have been a risk group since the beginning of the SARS‐CoV‐2 pandemic due to more significant complications, hospitalizations or deaths. The immunosuppressive state of cancer treatments or the tumor itself could influence the development of post‐vaccination antibodies. This study prospectively analyzed 89 patients under chemotherapy and/or immunotherapy, who received two doses of the mRNA‐1237 vaccine, and were compared with a group of 26 non‐cancer individuals. Information on adverse events and neutralizing antibodies against the ancestral strain of SARS‐CoV‐2 (WH1) have been analyzed. Local reactions accounted for 65%, while systemic reactions accounted for 46% of oncologic individuals/cancer patients. Regarding the response to vaccination, 6.7% of cancer patients developed low neutralizing antibody levels. Lower levels of neutralizing antibodies between cancer and non‐cancer groups were significant in individuals without previous SARS‐CoV‐2 infection, but not in previously infected individuals. We also observed that patients receiving chemotherapy or chemoimmunotherapy have significantly lower levels of neutralizing antibodies than non‐cancer individuals. In conclusion, our study confirms the importance of prioritizing cancer patients receiving anticancer treatment in SARS‐CoV‐2 vaccination programs.     

BRCA2 inhibition enhances cisplatin‐mediated alterations in tumor cell proliferation,metabolism, and metastasis

Tumor cells have unstable genomes relative to non‐tumor cells. Decreased DNA integrity resulting from tumor cell instability is important in generating favorable therapeutic indices, and intact DNA repair mediates resistance to therapy. Targeting DNA repair to promote the action of anti‐cancer agents is therefore an attractive therapeutic strategy. BRCA2 is involved in homologous recombination repair. BRCA2 defects increase cancer risk but, paradoxically, cancer patients with BRCA2 mutations have better survival rates. We queried TCGA data and found that BRCA2 alterations led to increased survival in patients with ovarian and endometrial cancer. We developed a BRCA2‐targeting second‐generation antisense oligonucleotide (ASO), which sensitized human lung, ovarian, and breast cancer cells to cisplatin by as much as 60%. BRCA2 ASO treatment overcame acquired cisplatin resistance in head and neck cancer cells, but induced minimal cisplatin sensitivity in non‐tumor cells. BRCA2 ASO plus cisplatin reduced respiration as an early event preceding cell death, concurrent with increased glucose uptake without a difference in glycolysis. BRCA2 ASO and cisplatin decreased metastatic frequency in vivo by 77%. These results implicate BRCA2 as a regulator of metastatic frequency and cellular metabolic response following cisplatin treatment. BRCA2 ASO, in combination with cisplatin, is a potential therapeutic anti‐cancer agent.     

Suppression of non‐small‐cell lung cancer A549 tumor growth by an mtDNA mutation‐targeting pyrrole‐imidazole polyamide‐triphenylphosphonium and a senolytic drug

Certain somatic mutations in mtDNA were associated with tumor progression and frequently found in a homoplasmic state. We recently reported that pyrrole‐imidazole polyamide conjugated with the mitochondria‐delivering moiety triphenylphosphonium (PIP‐TPP) targeting an mtDNA mutation efficiently induced apoptosis in cancer cells with the mutation but not normal cells. Here, we synthesized the novel PIP‐TPP, CCC‐021‐TPP, targeting ND6 14582A > G homoplasmic missense mutation that is suggested to enhance metastasis of non‐small‐cell lung cancer A549 cells. CCC‐021‐TPP did not induce apoptosis but caused cellular senescence in the cells, accompanied by a significant induction of antiapoptotic BCL‐XL. Simultaneous treatment of A549 cells with CCC‐021‐TPP and the BCL‐XL selective inhibitor A‐1155463 resulted in apoptosis induction. Importantly, the combination induced apoptosis and suppressed tumor growth in an A549 xenografted model. These results highlight the potential of anticancer therapy with PIP‐TPPs targeting mtDNA mutations to induce cell death even in apoptosis‐resistant cancer cells when combined with senolytics.     

Suppression of high mobility group box 1 in B16F10 tumor does not inhibit the induction of neoantigen‐specific T cells

Accumulated clinical data of immune checkpoint blockades have suggested the importance of neoantigens in cancer immunity. Tumor antigens are released from dead cancer cells together with cellular components, such as damage‐associated molecular patterns (DAMPs), into the tumor microenvironment. We recently reported that high mobility group box 1 (HMGB1), a representative DAMP molecule, showed a negative impact on anti‐tumor immunity. However, a positive role of HMGB1 in the initiation of innate and subsequent adaptive immunity has also been demonstrated; thus, the effects of HMGB1 on anti‐tumor immunity have not been well understood. In this study, we identified nine immunogenic neoantigen epitopes of B16F10 murine melanoma cells and subsequently investigated the effects of suppression of HMGB1 on the induction of neoantigen‐specific immunity using HMGB1‐knockout tumors. Neoantigen‐reactive T cells were expanded in B16F10 tumor‐bearing mice, and T cell receptor repertoire analysis suggested that neoantigen‐reactive T cells were oligo‐clonally increased in B16F10 tumor bearers. An increase of neoantigen‐reactive T cells and oligoclonal expansion of the T cells were similarly detected in HMGB1‐knockout tumor‐bearing mice. The induction of neoantigen‐specific immunity under the suppression of HMGB1 in the tumor microenvironment shown in this study supports further development of combination therapy of HMGB1 suppression with neoantigen‐targeted cancer immunotherapies, including immune checkpoint blockade therapy.     

Improving function of cytotoxic T‐lymphocytes by transforming growth factor‐β inhibitor in oral squamous cell carcinoma

Immunotherapy with immune‐checkpoint therapy has recently been used to treat oral squamous cell carcinomas (OSCCs). However, improvements in current immunotherapy are expected because response rates are limited. Transforming growth factor‐β (TGF‐β) creates an immunosuppressive tumor microenvironment (TME) by inducing the production of regulatory T‐cells (Tregs) and cancer‐associated fibroblasts and inhibiting the function of cytotoxic T‐lymphocytes (CTLs) and natural killer cells. TGF‐β may be an important target in the development of novel cancer immunotherapies. In this study, we investigated the suppressive effect of TGF‐β on CTL function in vitro using OSCC cell lines and their specific CTLs. Moreover, TGFB1 mRNA expression and T‐cell infiltration in 25 OSCC tissues were examined by in situ hybridization and multifluorescence immunohistochemistry. We found that TGF‐β suppressed the function of antigen‐specific CTLs in the priming and effector phases in vitro. Additionally, TGF‐β inhibitor effectively restored the CTL function, and TGFB1 mRNA was primarily expressed in the tumor invasive front. Interestingly, we found a significant negative correlation between TGFB1 mRNA expression and the CD8⁺ T‐cell/Treg ratio and between TGFB1 mRNA expression and the Ki‐67 expression in CD8⁺ T‐cells, indicating that TGF‐β also suppressed the function of CTLs in situ. Our findings suggest that the regulation of TGF‐β function restores the immunosuppressive TME to active status and is important for developing new immunotherapeutic strategies, such as a combination of immune‐checkpoint inhibitors and TGF‐β inhibitors, for OSCCs.     

Mitotic arrest and slippage induced by pharmacological inhibition of Polo‐like kinase 1

Exposure to drugs that interfere with microtubule dynamics block cell cycle progression at mitosis by prolonged activation of the spindle assembly checkpoint (SAC). Cells can evade mitotic arrest and proceed to interphase without chromosome segregation by a process termed mitotic slippage that involves Cyclin B1 degradation without checkpoint inactivation. Here, we explored the cellular response to small‐molecule inhibitors of Polo‐like kinase 1 (Plk1), an important regulator of cell division. We found that the clinical Plk1 inhibitors BI 2536 and BI 6727, both unexpectedly, induced a dose‐dependent cellular drug response: While mitotic arrest was induced in cancer cell lines and primary non‐transformed cells across the entire range of concentrations tested, only high concentrations seemed to promote mitotic slippage. Since this observation contrasts with the effects expected from studies reporting RNAi‐mediated Plk1 depletion in cancer cells, we wondered whether both ATP‐competitive inhibitors target unknown kinases that are involved in signaling from the spindle assembly checkpoint (SAC) and might contribute to the mitotic slippage. A chemical proteomics approach used to profile the selectivity of both inhibitors revealed that SAC kinases are not targeted directly. Still, the activities of Cdk1/Cyclin B1 and Aurora B, which plays important roles in the error correction of false microtubule‐kinetochore attachments and in checkpoint signaling, were shown to be downregulated at high inhibitor concentrations. Our data suggest that the inhibition of Plk1 activity below a certain threshold influences Aurora B activity via reduced phosphorylation of Fox M1 and Survivin leading to diminished levels of Aurora B protein and alteration of its subcellular localization. Within the spectrum of SAC proteins that are degraded during mitotic slippage, the degradation of Cyclin B1 and the downregulation of Aurora B activity by Plk1 inhibition seem to be critical promoters of mitotic slippage. The results indicate that careful dose‐finding studies in cancer trials are necessary to limit or even prevent mitotic slippage, which could be associated with improved cancer cell survival.     

Activation of β2‐adrenergic receptor signals suppresses mesenchymal phenotypes of oral squamous cell carcinoma cells

Metastasis is a primary reason related to the mortality of oral squamous cell carcinoma (OSCC) patients. A program called epithelial‐mesenchymal transition (EMT) has been shown to play a critical role in promoting metastasis in epithelium‐derived carcinoma. During EMT, epithelial cancer cells acquire motile mesenchymal phenotypes and detach from primary tumors. Recent lines of evidence have suggested that EMT confers cancer cells with tumor‐initiating ability. Therefore, selective targeting of EMT would lead to the development of effective therapeutic agents. In this study, using a chemical biology approach, we identified isoxsuprine, a β2‐adrenergic receptor (β2‐AR) agonist as a low‐molecular‐weight compound that interferes with the acquisition of mesenchymal phenotypes of oral cancer cells. Treatment of multiple types of oral cancer cells with isoxsuprine led to the downregulation of mesenchymal cell markers that was accompanied by reduced cell motility. Similar inhibitory effects were also observed for isoprenaline, a non‐selective β‐adrenergic receptor (β‐AR) agonist. In addition, inhibition of cell migration upon treatment with isoxsuprine was reverted by a non‐selective β‐AR antagonist, propranolol, and the CRISPR/Cas9 system‐mediated deletion of the β2‐AR gene, suggesting that the effects exerted by isoxsuprine involved signals mediated by β2‐AR. In addition, in a subcutaneous xenograft model of oral cancer cells, the administration of isoxsuprine effectively suppressed primary tumor growth, suggesting β2‐AR signals to be a promising cancer therapeutic target for treatment of OSCC.     

Combination Strategies on the Basis of Immune Checkpoint Inhibitors in Non–Small-Cell Lung Cancer: Where Do We Stand?

The era of immune checkpoint inhibitors, especially programmed death-1 (PD-1)/programmed death ligand-1 (PD-L1) antibodies in the treatment of advanced non–small-cell lung cancer (NSCLC) is coming. Because of the lack of the definite biomarkers to select the optimal responders, only approximately 20% of patients with advanced NSCLC would respond to single checkpoint inhibitors-based immunotherapy. Moreover, primary or acquired resistance to conventional therapies is inevitable in most cases. Thus, combinations are pushed to move forward to be an alternative strategy and surely, it would be a future direction. Combination approaches on the basis of PD-1/PD-L1 inhibitors are currently designed to re-energize the immune system with complementary/synergetic mechanisms and could achieve durable antineoplastic effects in NSCLC. Herein, we highlight the potential combinations on the basis of PD-1/PD-L1 inhibitors in NSCLC, with other immunotherapies, chemotherapy, radiotherapy, and targeted therapy in this current review.     

DDR1 functions as an immune negative factor in colorectal cancer by regulating tumor‐infiltrating T cells through IL‐18

Immunotherapies represented by programmed cell death protein 1/programmed cell death ligand 1 (PD‐1/PD‐L1) immune checkpoint inhibitors have made great progress in the field of anticancer treatment, but most colorectal cancer patients do not benefit from immunotherapy. Discoidin domain receptor 1 (DDR1), a tyrosine kinase receptor, is activated by collagen binding and overexpressed in various malignancies. However, the role of DDR1 in colorectal cancer and immunoregulation remains unclear. In this study, we found DDR1 is highly expressed in colorectal cancer tissues and negatively associated with patient survival. We demonstrated that DDR1 promotes colorectal tumor growth only in vivo. Mechanistically, DDR1 is a negative immunomodulator in colorectal cancer and is involved in low infiltration of CD4⁺ and CD8⁺ T cells by inhibiting IL‐18 synthesis. We also reported that DDR1 enhances the expression of PD‐L1 through activating the c‐Jun amino terminal kinase (JNK) signaling pathway. In conclusion, our findings elucidate the immunosuppressive role of DDR1 in colorectal cancer, which may represent a novel target to enhance the efficacy of immunotherapy in colorectal cancer.     

Trametinib overcomes KRAS‐G12V–induced osimertinib resistance in a leptomeningeal carcinomatosis model of EGFR‐mutant lung cancer

Leptomeningeal carcinomatosis (LMC) occurs frequently in non–small cell lung cancer (NSCLC) harboring epidermal growth factor receptor (EGFR) mutations and is associated with acquired resistance to EGFR tyrosine kinase inhibitors (EGFR‐TKIs). However, the mechanism by which LMC acquires resistance to osimertinib, a third‐generation EGFR‐TKI, is unclear. In this study, we elucidated the resistance mechanism and searched for a novel therapeutic strategy. We induced osimertinib resistance in a mouse model of LMC using an EGFR‐mutant NSCLC cell line (PC9) via continuous oral osimertinib treatment and administration of established resistant cells and examined the resistance mechanism using next‐generation sequencing. We detected the Kirsten rat sarcoma (KRAS)‐G12V mutation in resistant cells, which retained the EGFR exon 19 deletion. Experiments involving KRAS knockdown in resistant cells and KRAS‐G12V overexpression in parental cells revealed the involvement of KRAS‐G12V in osimertinib resistance. Cotreatment with trametinib (a MEK inhibitor) and osimertinib resensitized the cells to osimertinib. Furthermore, in the mouse model of LMC with resistant cells, combined osimertinib and trametinib treatment successfully controlled LMC progression. These findings suggest a potential novel therapy against KRAS‐G12V–harboring osimertinib‐resistant LMC in EGFR‐mutant NSCLC.     

Immune response drives outcomes in prostate cancer: implications for immunotherapy

The heterogeneity of the immune microenvironment leads to different responses in immune checkpoint blockade therapy. We aimed to propose a robust molecular classification system to investigate the relevance of the immune microenvironment subtype and prognosis of prostate cancer patients, as well as the therapeutic response to immune checkpoint blockade therapy. A total of 1,557 prostate cancer patients were enrolled, including 69 real‐world samples from our institute (titled the AHMU‐PC cohort). The non‐negative matrix factorization algorithm was employed to virtually microdissect patients. The immune enrichment was characterized by a high enrichment of T cell‐, B cell‐, NK cell‐, and macrophage‐associated signatures, by which patients were subclassified into nonimmune and immune classes. Subsequently, the immune class was dichotomized into immune‐activated and immune‐suppressed subtypes based on the stromal signature, represented by the activation of WNT/TGF‐β, TGF‐β1, and C‐ECM signatures. Approximately 14.9% to 24.3% of patients belonged to the immune‐activated subtype, which was associated with favorable recurrence‐free survival outcomes. In addition, patients in the immune‐activated subtype were predicted to benefit more from anti‐PD‐1/PD‐L1 therapy. In conclusion, our study identifies a novel immune molecular classifier that is closely related to clinical prognosis and provides novel insights into immunotherapeutic strategies for prostate cancer patients.     

CD24, not CD47, negatively impacts upon response to PD‐1/L1 inhibitors in non–small‐cell lung cancer with PD‐L1 tumor proportion score < 50

CD24, a heavily glycosylated glycosylphosphatidylinositol–anchored surface protein, inhibits phagocytosis as potently as CD47. The relationship between such anti‐phagocytic factors and the immune response with immune–checkpoint inhibitors (ICI) remains unexplored. We evaluated CD24 and CD47 tumor proportion scores (TPS) in 68 of the 106 patients with advanced non–small‐cell lung cancer who participated in a prospective observational study of ICI treatment. We also explored the impact of CD24 TPS and CD47 TPS on ICI efficacy and serum cytokine changes. CD24 positivity (TPS ≥ 1) was negatively associated with progression–free survival (PFS) of ICI when PD‐L1 TPS was < 50 (median PFS; 37 vs 127 d, P = .033), but there was no association when PD‐L1 TPS was ≥ 50 (median PFS; 494 vs 144 d, P = .168). CD24 positivity was also related to significantly higher increase of CCL2 from baseline to 4‐6 wk later, and such increase was notably observed only when PD‐L1 TPS < 50 (P = .0004). CCL2 increase after ICI initiation was negatively predictive for survival after initiation of ICI (median survival time; not reached vs 233 d; P = .028). CD47 TPS high (≥60) significantly suppressed the increase in vascular endothelial growth factor (VEGF)‐A, D and PDGF‐AB/BB after ICI initiation. There was no association, however, between CD47 tumor expression and the efficacy of ICI. In conclusion, CD24, not CD47, is a candidate negative predictive marker of ICI in advanced, non–small‐cell lung cancer with PD‐L1 TPS < 50. Tumor expression of both CD24 and CD47 was associated with changes in factors related to monocytes and angiogenesis after ICI initiation (UMIN000024414).