Immune checkpoint blockade (ICB) has demonstrated clinical efficacy in several cancers, including melanoma, lung, colorectal, and liver malignancies. However, a substantial proportion of patients fail to respond, underscoring the need for alternative immunotherapeutic strategies capable of overcoming resistance to conventional checkpoint inhibition. One such strategy involves targeting intracellular inhibitory immune checkpoints that regulate effector lymphocyte function. Rasal1, a Ras GTPase-activating protein, has been shown to negatively regulate T cell-mediated antitumor immunity. In this study, we further characterized the impact of Rasal1 impairment on tumor progression, T cell stemness, and effector function. Using an endonuclease-mediated mutation targeting the C2 domain of Rasal1, we demonstrate that Rasal1-impaired (Rasal1i) mice exhibit significantly reduced tumor growth across multiple murine cancer models. Rasal1i mice displayed increased intratumoral CD8+ T cell accumulation, activation, cytolytic capacity, and enhanced Wnt signaling. Tumor-infiltrating lymphocytes additionally exhibited increased progenitor and stem-like memory phenotypes. Notably, Rasal1 inhibition prolonged survival and potentiated aPD-1 therapy in a resistant PD-L1-expressing B16F10 melanoma model. Collectively, these findings identify Rasal1 as an intracellular inhibitory immune checkpoint that constrains T cell stemness and antitumor function, and support its further evaluation as a therapeutic target for cancer immunotherapy. ### Competing Interest Statement The authors have declared no competing interest. Canadian Institutes of Health Research, https://ror.org/01gavpb45

Regulatory T cells (Tregs) maintain immune homeostasis by suppressing excessive immune responses. In the context of cancer, Tregs are abundantly recruited to inhibit immunity against tumoral cells, facilitate immune evasion, and promote tumor progression. While Treg depletion strategies have repeatedly failed in the clinic due to severe autoimmune side effects, lack of specificity, and rapid compensatory recruitment, a critical unmet need remains for safer and more effective approaches. Emerging evidence highlights the remarkable plasticity of Tregs, allowing them to adopt an inflammatory phenotype in response to tumor-associated cytokines. Thus, leveraging this plasticity, rather than attempting broad depletion, may represent a superior anticancer strategy. This plasticity is marked by the expression of transcription factors like T-bet (Th1-like) and RORγt (Th17-like), the production of pro-inflammatory cytokines such as IFN-γ and IL-17, and even the acquisition of differential energetic preferences pertaining to glucose or glutamine. These changes can weaken Treg suppressive functions or paradoxically enhance inflammation in the tumor microenvironment, thereby creating a complex interplay between immune suppression and anti-tumor effector activity. Understanding the molecular cues driving Treg plasticity is therefore critical for designing novel therapies that shift Tregs toward an effector-like state, ultimately enhancing anti-tumor immunity and improving the efficacy of current immunotherapies. This review offers a fresh perspective on how Treg plasticity can be therapeutically harnessed to overcome the persistent limitations of conventional Treg-targeted approaches.

Despite enormous efforts being invested in the development of novel therapies for brain malignancies, there remains a dire need for effective treatments, particularly for pediatric glioblastomas. Their poor prognosis has been attributed to the fact that conventional therapies target tumoral cells, but not glioblastoma stem cells (GSCs). GSCs are characterized by self-renewal, tumorigenicity, poor differentiation, and resistance to therapy. These characteristics represent the fundamental tools needed to recapitulate the tumor and result in a relapse. The mechanisms by which GSCs alter metabolic cues and escape elimination by immune cells are discussed in this article, along with potential strategies to harness effector immune cells against GSCs. As cellular immunotherapy is making significant advances in a variety of cancers, leveraging this underexplored reservoir may result in significant improvements in the treatment options for brain malignancies.

Energetic and nutritional requirements play a crucial role in shaping the immune cells that infiltrate tumor and parasite infection sites. The dynamic interaction between immune cells and the microenvironment, whether in the context of tumor or helminth infection, is essential for understanding the mechanisms of immunological polarization and developing strategies to manipulate them in order to promote a functional and efficient immune response that could aid in the treatment of these conditions. In this review, we present an overview of the immune response triggered during tumorigenesis and establishment of helminth infections, highlighting the transition to chronicity in both cases. We discuss the energetic demands of immune cells under normal conditions and in the presence of tumors and helminths. Additionally, we compare the metabolic changes that occur in the tumor microenvironment and the infection site, emphasizing the alterations that are induced to redirect the immune response, thereby promoting the survival of cancer cells or helminths. This emerging discipline provides valuable insights into disease pathogenesis. We also provide examples of novel strategies to enhance immune activity by targeting metabolic pathways that shape immune phenotypes, with the aim of achieving positive outcomes in cancer and helminth infections.

Immune checkpoint blockade (ICB) of negative co-receptors on T-cells such as programmed cell death-1 (PD-1) is promising for the treatment of cancer. Despite success, the poor prognosis for most patients highlights the need for novel clinical interventions. We have shown that the kinase, glycogen synthase kinase-3 (GSK-3) negatively regulates T-cell activation due to altered PD-1 and LAG-3 expression (Taylor et al., 2016 Immunity; Rudd et al., 2019 Cell Reports). GSK-3 inhibition (GSK-3i) is as effective as anti-PD-1 in controlling the growth of melanoma (Taylor et al., 2017 Can Res; Krueger and Rudd, Immunity 2017; Stelle et al., 2021 iScience). GSK-3i increases Tbet (Tbx21) transcription, which inhibits PD-1/LAG-3 transcription, while increasing granzyme B (GZMB) and interferon gamma (IFNγ). Here, we show that Gsk3−/− mice and small molecule inhibitors (SMIs) synergize with anti-PD-1 to eliminate melanomas that are resistant to anti-PD-1 monotherapy. Transcriptomic profiling showed that GSK-3 × PD-1 cooperativity was characterized by a specific increase in a family of different granzymes (7/12 GZM genes out of a data base of 20,500 potential genes). Some GZMs have been characterized and others not, but as a family, this increased armory of GZMs in CD8+ T-cells is expected to greatly enhance tumor killing. Overall, our data shows the PD-1 × GSK-3 synergy in limiting tumor growth is due to a specific set of cytolysis mediators needed for tumor killing.

Melanomas commonly undergo a phenotype switch, from a proliferative to an invasive state. Such tumor cell plasticity contributes to immunotherapy resistance; however, the mechanisms are not completely understood and thus are therapeutically unexploited. Using melanoma mouse models, we demonstrated that blocking the MNK1/2-eIF4E axis inhibited melanoma phenotype switching and sensitized melanoma to anti-PD-1 immunotherapy. We showed that phospho-eIF4E-deficient murine melanomas expressed high levels of melanocytic antigens, with similar results verified in patient melanomas. Mechanistically, we identified phospho-eIF4E-mediated translational control of NGFR, a critical effector of phenotype switching. Genetic ablation of phospho-eIF4E reprogrammed the immunosuppressive microenvironment, exemplified by lowered production of inflammatory factors, decreased PD-L1 expression on dendritic cells and myeloid-derived suppressor cells, and increased CD8+ T cell infiltrates. Finally, dual blockade of the MNK1/2-eIF4E axis and the PD-1/PD-L1 immune checkpoint demonstrated efficacy in multiple melanoma models regardless of their genomic classification. An increase in the presence of intratumoral stem-like TCF1+PD-1+CD8+ T cells, a characteristic essential for durable antitumor immunity, was detected in mice given a MNK1/2 inhibitor and anti-PD-1 therapy. Using MNK1/2 inhibitors to repress phospho-eIF4E thus offers a strategy to inhibit melanoma plasticity and improve response to anti-PD-1 immunotherapy.

Multiple myeloma (MM) is a hematological cancer in which relapse and resistance are highly frequent. Therefore, alternatives to conventional treatments are necessary. Withaferin A, a withanolide isolated from Withania somnifera, has previously shown promising activity against various MM models. In the present study, structure-activity relationships (SARs) were evaluated using 56 withanolides. The antiproliferative activity was assessed in three MM cell lines and in a 3D MM coculture model to understand the in vitro activity of compounds in models of various complexity. While the results obtained in 2D allowed a quick and simple evaluation of cytotoxicity used for a first selection, the use of the 3D MM coculture model allowed filtering compounds that perform better in a more complex setup. This study shows the importance of the last model as a bridge between 2D and in vivo studies to select the most active compounds and ultimately lead to a reduction of animal use for more sustained in vivo studies. NF-κB inhibition was determined to evaluate if this could be one of the targeted pathways. The most active compounds, withanolide D (2) and 38, should be further evaluated in vivo.

Immunotherapy using checkpoint blockade (ICB) with antibodies such as anti-PD-1 has revolutionised the treatment of many cancers. Despite its use to treat COVID-19 patients and autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis, the effect of hydroxychloroquine (HCQ) on cancer immunotherapy has not been examined. In this study, remarkably, we find that HCQ alone, or in combination with azithromycin (AZ), at doses used to treat patients, decreased the therapeutic benefit of anti-PD-1 in cancer immunotherapy. No deleterious effect was seen on untreated tumors. Mechanistically, HCQ and HCQ/AZ inhibited PD-L1 expression on tumor cells, while specifically targeting the anti-PD-1 induced increase in progenitor CD8+CD44+PD-1+TCF1+ tumor infiltrating T cells (TILs) and the generation of CD8+CD44+PD-1+ effectors. Surprisingly, it also impaired the appearance of a subset of terminally exhausted CD8+ TILs. No effect was seen on the presence of CD4+ T cells, FoxP3+ regulatory T cells (Tregs), thymic subsets, B cells, antibody production, myeloid cells, or the vasculature of mice. This study indicates for the first time that HCQ and HCQ/AZ negatively impact the ability of anti-PD-1 checkpoint blockade to promote tumor rejection.

The serine/threonine kinase, glycogen synthase kinase 3 (GSK-3) has been implicated in immune cell activation and function. Our recent studies have shown that the abrogation of GSK-3 activity down-regulates the expression of key inhibitory receptors PD-1 and LAG-3. It also regulates the expression of the transcription factor NFAT which, in turn, is responsible for inhibiting PD-1/LAG-3 transcription as well as activating the expression of cytolytic effector proteins such as perforin and granzyme B. The role of components of the Wnt signaling pathway in these events remains to be fully uncovered. This mini-review discusses the recent discoveries that have elucidated the role of the GSK-3 signaling pathway in cancer immunotherapy.

Coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a serious threat to global public health. Hydroxychloroquine (HCQ) and the antibiotic azithromycin (AZ) are still being used by thousands and numerous hospitals to treat COVID-19. In a related context, immunotherapy using checkpoint blockade (ICB) with antibodies such as anti-PD-1 has revolutionised cancer therapy. Given that cancer patients on ICB continue to be infected with SARS-CoV-2, an understanding of the effects of HCQ and AZ on the elimination of tumors by anti-PD-1 ICB is urgently needed. In this study, we report that HCQ alone, or in combination with AZ, at doses used to treat COVID-19 patients, reverses the therapeutic benefit of anti-PD-1 in controlling B16 melanoma tumor growth in mice. No deleterious effect was seen on untreated tumors, or in using AZ alone in anti-PD-1 immunotherapy. Mechanistically, HCQ and HCQ/AZ inhibited PD-L1 expression on tumor cells, while specifically targeting the anti-PD-1 induced increase in progenitor CD8+CD44+PD-1+TCF1+ tumor-infiltrating T-cells (TILs) and the generation of CD8+CD44+PD-1+ effectors. Surprisingly, it also blocked the appearance of a subset of terminally exhausted CD8+ TILs. No effect was seen on the presence of CD4+ T-cells, FoxP3+ Tregs, thymic subsets, B-cells, antibody production, myeloid cells, or the vasculature of mice. Lastly, we identified TCF-1 expression in peripheral CD8+ T-cells from cancer or non-cancer human patients infected with SARs CoV2 as a marker for the effects of COVID-19 and HCQ on the immune system. This study indicates for the first time that HCQ and HCQ/AZ negatively impact the ability of anti-PD-1 checkpoint blockade to promote tumor rejection.

Multiple myeloma is a hematological cancer characterized by the clonal proliferation of malignant plasma cells in the bone marrow. That disease has a rather low incidence but displays a high rate of relapse and resistance to conventional therapies. It is therefore necessary to find new therapeutic strategies to overcome this resistance, which is partly attributed to a subpopulation of cells known as cancer stem cells. Withanolides and HDAC6 selective inhibitors were identified as promising compounds in various resistant multiple myeloma models.

IntroductionBreast cancer is the second leading cause of death due to cancer in women, and often becomes multidrug resistant (MDR) to adjuvant drug therapies due to the overexpression of ATP‐binding cassette (ABC) drug efflux transporters or through the decreased expression of drug uptake transporters, which can lead to treatment failure. MDR breast cancer cells can have complex drug translocation processes due to attempts to reduce intracellular anti‐cancer drug concentrations. It is critical to investigate possible drug targets for MDR cancers that can lead to increased drug accumulation. Solute carrier organic anion (SLCO) transporters are a family of transmembrane proteins that can uptake amphiphilic organic compounds into cells. Preliminary evidence suggests that SLCO transporters may be responsible for the targeted uptake of an emerging class of anticancer molecules called jadomycins, topoisomerase II and aurora B kinase‐targeting compounds which retain their cytotoxic potency in ABC‐transporter overexpressing MDR breast cancer cells.ObjectiveThe objective of this study is to evaluate the gene expression of SLCO transporters in a panel of breast cancer cell subtypes.MethodsThe mRNA expression 11 SLCO transporters was quantified using quantitative polymerase chain reaction (qPCR) in drug sensitive MCF7 (MCF7‐CON) and taxol (MCF7‐TXL), etoposide (MCF7‐ETP), and mitoxantrone (MCF7‐MITX) resistant MCF7 breast cancer cell lines which overexpress ABCB1, ABCC1, and ABCG2 drug efflux transporters, respectively. Comparisons were also made between BT474, SKBR3, and MDA‐MB‐231 breast cancer cell lines with different hormone receptor profiles as well as non‐cancerous MCF‐10A breast epithelial cells.ResultsGenerally, SLCO4A1 and 3A1 are significantly higher than the remaining transporters in drug resistant MCF7 cells, whereas this is not seen in the drug sensitive MCF7‐CON cells. There was significantly higher expression of: SLCO3A1 and 4A1 compared to the remaining transporters in MCF7‐MITX cells; SLCO3A1, 4A1, 4C1 compared to the remaining transporters in MCF7‐TXL cells; SLCO4A1 compared to the remaining transporters in MCF7‐ETP and SKBR3 cells; and SLCO3A1 and 4C1 versus all other transporters in MCF7‐ETP cells. There was significantly higher expression of SLCO1C1 versus SLCO3A1, 5A1, 1B3, 2B1, 1B1, 2A1, and 6A1 in MCF7‐CON cells.When differentiating between cancerous cells and non‐transformed MCF‐10A cells there were no significant differences between SLCO3A1 and any transporters found in breast epithelial MCF‐10A cells, however there was significantly higher expression of SLCO4A1 versus SLCO5A1, 1B3, 2B1, 1B1, and 4C1. Furthermore, there were no significant differences when comparing the transporters in BT474 and MDA‐MB‐231 cell lines.ConclusionSLCOs are differently expressed in breast cancer cell lines. SLCO4A1 and 3A1 were the most commonly expressed at significantly higher levels versus the other transporter genes in the cell lines used. They also were most highly expressed in the MDR MCF7 breast cancer cell lines and could serve as a conserved transport mechanism for intracellular delivery of anti‐cancer drugs, thereby suggesting they may be responsible for the cellular uptake of jadomycins. To explore this hypothesis, our future work will evaluate the jadomycin accumulation and cytotoxicity in SLCO3A1 and 4A1 knockdown cell lines.Support or Funding InformationLeah Bennett is a trainee in the Cancer Research Training Program of the Beatrice Hunter Cancer Research Institute, with funds provided by a CIBC Graduate Scholarship in Medical Research and the QEll Foundation.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The effects of genistein on angiogenesis remain poorly understood. Some studies claim an antiangiogenic effect and others claim a pro-angiogenic one. Thus, the aim of this study was to determine if genistein may exhibit bivalent angiogenic effects. To address this question, genistein angiogenic modulatory effects were examined using an in vitro 3D angiogenesis model using human umbilical vein endothelial cells. In this model, a bivalent effect of genistein was demonstrated on sprouting angiogenesis, with angiogenic stimulation at low concentrations (0.001-1 mu M) and inhibition at higher ones (25-100 mu M). Enhancement of the endothelial tube formation correlated with an increase in human umbilical vein endothelial cell metabolic activity and proliferation. Inhibition of angiogenesis correlated with a decreased metabolic activity, proliferation, and migration. Moreover, high concentrations of genistein influenced human umbilical vein endothelial cell morphology. Expression of genes involved in the angiogenic process in response to genistein was measured to study the mechanism of action. Secretome profiling revealed that angiogenic regulators were modulated with genistein treatment. These results suggested a bivalent effect of genistein on human umbilical vein endothelial cell growth and angiogenesis, and further investigations on the benefit of genistein for cancer chemoprevention, cancer treatment, or pro-angiogenic therapies have to be carefully considered.

Multiple myeloma is characterized by the accumulation of malignant plasma cells in the bone marrow. Multiple myeloma is the second most frequently diagnosed hematological malignancy, predominantly affecting the elderly. Despite recent advances in the development of novel therapies, multiple myeloma remains an incurable malignancy where the majority of patients relapse, develop resistance, and eventually die from the disease. This has been attributed to the fact that conventional therapy currently in use targets mainly the bulk of tumor cells, but not the tumor-initiating cancer stem cells. Cancer stem cells are a highly resistant subpopulation of cells believed to be responsible for the initiation, progression, metastasis, and relapse of cancer. Enormous efforts have been invested in the characterization of cancer stem cells. These efforts led to the characterization of key cellular signaling pathways responsible for conferring stem cell characteristics including self-renewal, differentiation, migratory, survival, and intracellular detoxification capabilities. Targeting these protective mechanisms offers a valuable strategy that may help combat a major driving force behind cancers. The use of natural products offers a promising therapeutic approach for targeting cancer stem cells. In this review, recent advances achieved in the characterization of cancer stem cells derived from hematological malignancies, with a particular focus on multiple myeloma, are discussed and major natural products that target cancer stem cells are presented. As natural products remain an essential source of novel chemical structures and medicinal leads, the exploitation of this immense reservoir is used to draw lessons in targeting multiple myeloma-cancer stem cells.

Little is known about the biological and structural features that govern the isoform selectivity for class I histone deacetylases (HDACs) over HDAC6. In addition to that for known inhibitors, like benzamides, psammaplin A, and cyclodepsipeptide-derived thiols, selectivity was also observed for naturally occurring cyclopeptide HDAC inhibitors with an aliphatic flexible linker and ketonelike zinc-binding group (ZBG). The present study reports that this isoform selectivity is mainly due to the linker and ZBG, as replacement of the cyclopeptide cap region by a simple aniline retained class I HDAC isoform selectivity toward HDAC6 in enzymatic assays. The best cyclopeptide-free analogues preserved efficacy against Plasmodium falciparum and cancer cell lines. Molecular modeling provided hypotheses to explain this selectivity and suggests different behaviors of the flexible linker on HDAC1 and HDAC6 pockets, which may influence, on the basis of the strength of the ZBG, its coordination with the zinc ion.

Multiple myeloma (MM) is a hematological malignancy, which remains incurable because most patients eventually relapse or become refractory to current treatments. Due to heterogeneity within the cancer cell microenvironment, cancer cell populations employ a dynamic survival strategy to chemotherapeutic treatments, which frequently results in a rapid acquisition of therapy resistance. Besides resistance-conferring genetic alterations within a tumor cell population selected during drug treatment, recent findings also reveal non-mutational mechanisms of drug resistance, involving a small population of “cancer stem cells” (CSCs) which are intrinsically more refractory to the effects of a variety of anticancer drugs. Other studies have implicated epigenetic mechanisms in reversible drug tolerance to protect the population from eradication by potentially lethal exposures, suggesting that acquired drug resistance does not necessarily require a stable heritable genetic alteration. Clonal evolution of MM cells and the bone marrow microenvironment changes contribute to drug resistance. MM-CSCs may not be a static population and survive as phenotypically and functionally different cell types via the transition between stem-like and non-stem-like states in local microenvironments, as observed in other types of cancers. Targeting MM-CSCs is clinically relevant, and different approaches have been suggested to target molecular, metabolic and epigenetic signatures, and the self-renewal signaling characteristic of MM CSC-like cells. Here, we summarize epigenetic strategies to reverse drug resistance in heterogeneous multiple myeloma.

Morphine is administered intravenously for pain management in the perioperative period. The effect of the inflammatory response to surgery on morphine distribution across the blood-brain barrier (BBB) in humans was investigated. We hypothesized that a graded surgically induced, systemic inflammatory response alters cerebrospinal fluid (CSF) levels of morphine, morphine-3-glucuronide (M3G), and morphine-6-glucuronide (M6G) through a temporary reduction in BBB drug efflux transporter function.

In spite of recent therapeutic advances, multiple myeloma (MM) remains a malignancy with very low curability. This has been partly attributed to the existence of a drug-resistant subpopulation known as cancer stem cells (CSCs). MM-CSCs are equipped with the necessary tools that render them highly resistant to virtually all conventional therapies. In this study, the growth inhibitory effects of withanolide D (WND), a steroidal lactone isolated from Withania somnifera, on drug-sensitive tumoral plasma cells and drug-resistant MM cells have been investigated. In MTT/XTT assays, WND exhibited similar cytostatic effects between drug-resistant and drug-sensitive cell lines in the nM range. WND also induced cell death and apoptosis in MM-CSCs and RPMI 8226 cells, as examined by the calcein/ethidium homodimer and annexin V/propidium iodide stainings, respectively. To determine whether P-glycoprotein (P-gp) efflux affected the cytostatic activity of WND, P-gp was inhibited with verapamil and results indicated that the WND cytostatic effect in MM-CSCs was independent of P-gp efflux. Furthermore, WND did not increase the accumulation of the fluorescent P-gp substrate rhodamine 123 in MM-CSCs, suggesting that WND may not inhibit P-gp at the tested relevant doses. Therefore, the WND-induced cytostatic effect may be independent of P-gp efflux. These findings warrant further investigation of WND in MM-CSC animal models.

Multiple myeloma (MM) remains an incurable malignancy despite the development of novel therapeutics. This is believed to be due to a subset of rare chemotherapy-resistant cancer stem cells (CSCs). Differentiation therapy represents one strategy aimed at reducing the stemness of CSCs. The anticancer effect of withaferin A (WFA) was studied in MM-CSCs and RPMI 8226 MM tumoral plasma cells (RPMIs). WFA exhibited growth inhibitory effects in both MM-CSCs and RPMIs, with IC50 values of 649 and 224 nM, respectively. WFA also induced a G2 cell cycle arrest, as well as cell death and apoptosis. Although, WFA did not exhibit a direct anti-migratory effect, a remarkable morphological change was observed in MM-CSCs in response to WFA treatment. Using qPCR gene expression analyses, WFA caused a reduction in stemness markers, and a promotion of differentiation markers in MM-CSCs. These results warrant further investigation of WFA in relevant MM animal models.