Tackling of Immunorefractory Tumors by Targeting Alternative Immune Checkpoints.

Physiologically, well known or traditional immune checkpoints (ICs), such as CTLA-4 and PD-1, are in place to promote tolerance to self-antigens and prevent generation of autoimmunity. In cancer, the ICs are effectively engaged by the tumor cells or stromal ells from the tumor microenvironment through expression of cognate ligands for the ICs present on the cell surface of CD8+ T lymphocytes. The ligation of ICs on CD8+ T lymphocytes triggers inhibitory signaling pathways, leading to quiescence or an exhaustion of CD8+ T lymphocytes. This results in failure of immunotherapy. To overcome this, several FDA-approved therapeutic antibodies are available, but the clinical outcome is quite variable due to the resistance encountered through upregulated expression of alternate ICs such as VISTA, LAG-3, TIGIT and TIM-3. This review focuses on the roles played by the traditional as well as alternate ICs and the contribution of associated signaling pathways in generating such resistance to immunotherapy. Combinatorial targeting of traditional and alternate ICs might be beneficial for immune-refractory tumors.

Ethnic disparities in the immune microenvironment of triple negative breast cancer and its role in therapeutic outcomes.

In 2020, newly diagnosed breast cancer (BC) cases surpassed that of lung cancer among women, making it the most common female cancer globally. In spite of recent increases in incidence rates, mortality due to BC has declined since 1989. These declines have been attributed to advancements in treatment modalities as well as increased mammography surveillance. Despite these advances, African American (AA) women are 40% more likely to die from BC than Caucasian women. Multifactorial etiology has been implicated in the disparity of BC mortality rates among AA women. As an example, AA women have a disproportionate incidence of triple negative breast cancer (TNBC), which has a poor prognosis and marginal treatment options. Increasingly, the tumor microenvironment (TME) has gained relevance as it relates to primary tumor progression, metastasis and treatment possibilities. The treatment outcomes or pathological complete response (pCR) in TNBC among AA women are affected by differences in TME. The TME of AA women exhibit several variances in acellular and cellular components associated with pro-tumorigenic effects. For example, increased levels of the adipocyte-related hormone, resistin, the pro-inflammatory cytokine, IL-6, and the CC chemokine, CCL2, within the TME of AA women gives rise to an increased density of M2 macrophages, also known as tumor-associated macrophages. Elevated levels of vascular endothelial growth factor in the TME of AA women increase the vascular density or vascularity, which facilitate aggressive tumor growth and metastasis. Furthermore, a pro-tumorigenic TME is supported by increased levels of the CXC chemokine, CXCL12 that results in the recruitment of regulatory T lymphocytes (Tregs ). Due to these and other differences in the TME of AA women, precision oncology can target specific aspects of the TME that may contribute to a poorer prognosis. In addition to the discrepancies in the TME, AA women face socio-economic barriers that limit their ability to access state-of-the-art, novel therapies against metastatic TNBC. In this review, we will provide a brief overview of the tumor immune microenvironment, immune-based treatment options for TNBC and their potential to decrease health disparities due to ethnicity.

LASP1 in Cellular Signaling and Gene Expression: More than Just a Cytoskeletal Regulator.

LIM and SH3 protein 1 was originally identified as a structural cytoskeletal protein with scaffolding function. However, recent data suggest additional roles in cell signaling and gene expression, especially in tumor cells. These novel functions are primarily regulated by the site-specific phosphorylation of LASP1. This review will focus on specific phosphorylation-dependent interaction between LASP1 and cellular proteins that orchestrate primary tumor progression and metastasis. More specifically, we will describe the role of LASP1 in chemokine receptor, and PI3K/AKT signaling. We outline the nuclear role for LASP1 in terms of epigenetics and transcriptional regulation and modulation of oncogenic mRNA translation. Finally, newly identified roles for the cytoskeletal function of LASP1 next to its known canonical F-actin binding properties are included.

IND-2, a Quinoline Derivative, Inhibits the Proliferation of Prostate Cancer Cells by Inducing Oxidative Stress, Apoptosis and Inhibiting Topoisomerase II.

In men, prostate cancer (PC) is the most frequently diagnosed cancer, causing an estimated 375,000 deaths globally. Currently, existing therapies for the treatment of PC, notably metastatic cases, have limited efficacy due to drug resistance and problematic adverse effects. Therefore, it is imperative to discover and develop novel drugs for treating PC that are efficacious and do not produce intolerable adverse or toxic effects. Condensed quinolines are naturally occurring anticancer compounds. In this study, we determined the in vitro efficacy of IND-2 (4-chloro-2-methylpyrimido[1″,2″:1,5]pyrazolo[3,4-b]quinolone) in the PC lines, PC-3 and DU-145. IND-2 significantly inhibited the proliferation of PC-3 and DU-145, with IC50 values of 3 µM and 3.5 µM, respectively. The incubation of PC-3 cells with 5 and 10 µM of IND-2 caused the loss of the mitochondrial membrane potential in PC-3 cells. Furthermore, IND-2, at 5 µM, increased the expression of cleaved caspase-3, cleaved caspase-7 and cleaved poly (ADP-ribose) polymerase (PARP). The incubation of PC-3 cells with 5 µM of IND-2 significantly decreased the expression of the apoptotic protein, B-cell lymphoma 2 (Bcl-2). Furthermore, 5 and 10 µM of IND-2 produced morphological changes in PC-3 cells characteristic of apoptosis. Interestingly, IND-2 (2.5, 5 and 10 µM) also induced mitotic catastrophe in PC-3 cells, characterized by the accumulation of multinuclei. The incubation of DU-145 cells with 1.25 and 5 µM of IND-2 significantly increased the levels of reactive oxygen species (ROS). Finally, IND-2, at 10 µM, inhibited the catalytic activity of topoisomerase IIα. Overall, our findings suggest that IND-2 could be a potential lead compound for the development of more efficacious compounds for the treatment of PC.

Role of eIF4A1 in triple-negative breast cancer stem-like cell-mediated drug resistance.

In cap-dependent translation, the eukaryotic translation initiation factor 4A (eIF4A1) is an mRNA helicase is involved in unwinding of the secondary structure, such as the stem-loops, at the 5'-leader regions of the key oncogenic mRNAs. This facilitates ribosomal scanning and translation of the oncogenic mRNAs. eIF4A1 has a regulatory role in translating many oncoproteins that have vital roles in several steps of metastases. Sridharan et. al. have discovered and provide a novel insight into how eIF4A1 can play a regulatory role in drug resistance by influencing the levels of pluripotent Yamanaka transcription factors and ATP-binding cassette (ABC) transporters in triple-negative breast cancer (TNBC) stem-like cells. These findings may help us understand the molecular underpinnings of chemoresistance, especially in established metastases in TNBC. Importantly, eIF4A1 may form a novel clinical target in metastatic TNBC and the drug eFT226 from Effector Therapeutics targeting eIF4A1 is already in phase1-2 clinical trial.

A Novel Dialkylamino-Functionalized Chalcone, DML6, Inhibits Cervical Cancer Cell Proliferation, In Vitro, via Induction of Oxidative Stress, Intrinsic Apoptosis and Mitotic Catastrophe.

In this study, we designed, synthesized and evaluated, in vitro, novel chalcone analogs containing dialkylamino pharmacophores in the cervical cancer cell line, OV2008. The compound, DML6 was selective and significantly decreased the proliferation of OV2008 and HeLa cells in sub-micromolar concentrations, compared to prostate, lung, colon, breast or human embryonic kidney cell line (HEK293). DML6, at 5 µM, arrested the OV2008 cells in the G2 phase. Furthermore, DML6, at 5 µM, increased the levels of reactive oxygen species and induced a collapse in the mitochondrial membrane potential, compared to OV2008 cells incubated with a vehicle. DML6, at 5 µM, induced intrinsic apoptosis by significantly (1) increasing the levels of the pro-apoptotic proteins, Bak and Bax, and (2) decreasing the levels of l the anti-apoptotic protein, Bcl-2, compared to cell incubated with a vehicle. Furthermore, DML6, at 5 and 20 µM, induced the cleavage of caspase-9, followed by subsequent cleavage of the executioner caspases, caspase-3 and caspase-7, which produced OV2008 cell death. Overall, our data suggest that DML6 is an apoptosis-inducing compound that should undergo further evaluation as a potential treatment for cervical cancer.

Alternative approaches to overcome chemoresistance to apoptosis in cancer.

Apoptosis, or programmed cell death, is a form of regulated cell death (RCD) that is essential for organogenesis and homeostatic maintenance of normal cell populations. Apoptotic stimuli activate the intrinsic and/or extrinsic pathways to induce cell death due to perturbations in the intracellular and extracellular microenvironments, respectively. In patients with cancer, the induction of apoptosis by anticancer drugs and radiation can produce cancer cell death. However, tumor cells can adapt and become refractory to apoptosis-inducing therapies, resulting in the development of clinical resistance to apoptosis. Drug resistance facilitates the development of aggressive primary tumors that eventually metastasize, leading to therapy failure and mortality. To overcome the resistance to apoptosis to neoadjuvant chemotherapy or targeted therapy, alternative targets of RCD can be induced in apoptosis-resistant cancer cells. Alternatively, cell death can be independent of apoptosis and this strategy could be utilized to develop novel anti-cancer therapies. This chapter discusses approaches that could be employed to overcome clinical resistance to apoptosis in cancer cells.

A Novel Thienopyrimidine Analog, TPH104, Mediates Immunogenic Cell Death in Triple-Negative Breast Cancer Cells.

Enhancing the tumor immunogenic microenvironment has been suggested to circumvent triple-negative breast cancer (TNBC) resistance and increase the efficacy of conventional chemotherapy. Here, we report a novel chemotherapeutic compound, TPH104, which induces immunogenic cell death in the TNBC cell line MDA-MB-231, by increasing the stimulatory capacity of dendritic cells (DCs), with an IC50 value of 140 nM. TPH104 (5 µM) significantly increased ATP levels in the supernatant and mobilized intracellular calreticulin to the plasma membrane in MDA-MB-231 cells, compared to cells incubated with the vehicle. Incubating MDA-MB-231 cells for 12 h with TPH104 (1-5 µM) significantly increased TNF-α mRNA levels. The supernatants of dying MDAMB-231 cells incubated with TPH104 increased mouse bone marrow-derived DC maturation, the expression of MHC-II and CD86 and the mRNA expression of TNF-α, IL-6 and IL-12. Overall, these results indicate that TPH104 induces immunogenic cell death in TNBC cells, in part, by activating DCs.

The CXCR4-Dependent LASP1-Ago2 Interaction in Triple-Negative Breast Cancer.

The CXCR4-LASP1 axis is an emerging target in the field of breast cancer metastasis. C-X-C chemokine receptor type 4 (CXCR4) mediates directed cell migration when activated by its cognate ligand CXCL12. LIM and SH3 Protein 1 (LASP1) is a critical node in the CXCR4 signaling pathway, as its deficiency blocks CXCR4-dependent Matrigel invasion. The mechanism by which LASP1 facilitates this invasive ability of tumor cells when CXCR4 is activated is unknown. Our previous proteomics work had revealed several components of the RNA interference (RNAi) machinery as being potential LASP1 interacting proteins. Here we report that argonaute 2 (Ago2), a protein with central involvement in RNAi, associates with LASP1 in triple-negative breast cancer (TNBC) cells. We demonstrate that LASP1 co-immunoprecipitates with Ago2 endogenously in a CXCL12-dependent manner, with further confirmation of this interaction by proximity ligation assay. Furthermore, this association is specific to CXCR4 as it can be abrogated by the CXCR4 antagonist, AMD3465. By GST-pulldown approach, we identify that LASP1 directly binds to Ago2 through its LIM and SH3 domains, and that this binding is dictated by the S146 and Y171 phosphorylation sites of LASP1. Additionally, the phosphorylation status of LASP1 affected tumor suppressor microRNA (miRNA) Let-7a-guided Ago2 activity. Levels of several endogenous targets of Let-7a were found to be altered including C-C chemokine receptor type 7 (CCR7), which is another critical chemokine receptor involved in metastasis to lymph nodes. Our results suggest a novel role for the LASP1-Ago2 module in shaping the RNAi landscape, functionally impacting the invasive ability of cancer cells.

Identification of Cardiac Glycosides as Novel Inhibitors of eIF4A1-Mediated Translation in Triple-Negative Breast Cancer Cells.

The eukaryotic translation initiation factor 4F complex (eIF4F) is a potential chemotherapeutic target in triple-negative breast cancer (TNBC). This complex regulates cap-dependent translational initiation and consists of three core proteins: eIF4E, eIF4G, and eIF4A1. In this study, we focus on repositioning compounds as novel inhibitors of eIF4A1-mediated translation. In order to accomplish this goal, a modified synthetic reporter assay was established. More specifically, a (CGG)4 motif, which confers eIF4A dependency, was incorporated into the 5'-leader region of a luciferase-tdTomato lentiviral reporter construct. The Prestwick Chemical Library was then screened in multiple TNBC cell lines by measuring the tdTomato fluorescent intensity. We identified several cardiac glycosides as potential inhibitors of eIF4A1-mediated translation. Based on our studies, we find that cardiac glycosides inhibit the expression of eIF4A1. To identify a potential mechanism by which this was occurring, we utilized the Integrative Library of Integrated Network-Based Cellular Signatures (iLINCS). Our pursuits led us to the discovery that cardiac glycosides also decrease levels of c-MYC. Quantitative PCR confirmed that decreases in c-MYC and eIF4A were occurring at the transcriptional level. As such, disruption of the eIF4A1-c-MYC axis may be a viable approach in the treatment of TNBC. The novel combination of rocaglamide A and digoxin exhibited synergistic anti-cancer activity against TNBC cells in vitro. The findings in this study and others are important for formulating potential combination chemotherapies against eIF4A1 in vivo. Thus, drug repositioning may be one classical approach to successfully target eIF4A1 in TNBC patients.

Role of the CXCR4-LASP1 Axis in the Stabilization of Snail1 in Triple-Negative Breast Cancer.

The CXCL12-CXCR4 axis plays a vital role in many steps of breast cancer metastasis, but the molecular mechanisms have not been fully elucidated. We previously reported that activation of CXCR4 by CXCL12 promotes the nuclear localization of LASP1 (LIM and SH3 protein 1). The nuclear LASP1 then interacts with Snail1 in triple-negative breast cancer (TNBC) cell lines. In this study, we report that the nuclear accumulation and retention of Snail1 was dependent on an increase in nuclear LASP1 levels driven by active CXCR4. The CXCR4-LASP1 axis may directly regulate the stabilization of nuclear Snail1, by upregulating nuclear levels of pS473-Akt, pS9-GSK-3ß, A20, and LSD1. Furthermore, the activation of CXCR4 induced association of LASP1 with Snail1, A20, GSK-3ß, and LSD1 endogenously. Thus, nuclear LASP1 may also regulate protein-protein interactions that facilitate the stability of Snail1. Genetic ablation of LASP1 resulted in the mislocalization of nuclear Snail1, loss of the ability of TNBC cells to invade Matrigel and a dysregulated expression of both epithelial and mesenchymal markers, including an increased expression of ALDH1A1, a marker for epithelial breast cancer stem-like cells. Our findings reveal a novel role for the CXCR4-LASP1 axis in facilitating the stability of nuclear localized Snail1.

Targeting of the Eukaryotic Translation Initiation Factor 4A Against Breast Cancer Stemness.

Breast cancer stem cells (BCSCs) are intrinsically chemoresistant and capable of self-renewal. Following chemotherapy, patients can develop minimal residual disease due to BCSCs which can repopulate into a relapsed tumor. Therefore, it is imperative to co-target BCSCs along with the bulk tumor cells to achieve therapeutic success and prevent recurrence. So, it is vital to identify actionable molecular targets against both BCSCs and bulk tumor cells. Previous findings from our lab and others have demonstrated that inhibition of the emerging drug target eIF4A with Rocaglamide A (RocA) was efficacious against triple-negative breast cancer cells (TNBC). RocA specifically targets the pool of eIF4A bound to the oncogenic mRNAs that requires its helicase activity for their translation. This property enables specific targeting of tumor cells. The efficacy of RocA against BCSCs is unknown. In this study, we postulated that eIF4A could be a vulnerable node in BCSCs. In order to test this, we generated a paclitaxel-resistant TNBC cell line which demonstrated an elevated level of eIF4A along with increased levels of cancer stemness markers (ALDH activity and CD44), pluripotency transcription factors (SOX2, OCT4, and NANOG) and drug transporters (ABCB1, ABCG2, and ABCC1). Furthermore, genetic ablation of eIF4A resulted in reduced expression of ALDH1A1, pluripotency transcription factors and drug transporters. This pointed out that eIF4A is likely associated with selected set of proteins that are critical to BCSCs, and hence targeting eIF4A may eliminate BCSCs. Therefore, we isolated BCSCs from two TNBC cell lines: MDA-Bone-Un and SUM-159PT. Following RocA treatment, the self-renewal ability of the BCSCs was significantly reduced as determined by the efficiency of the formation of primary and secondary mammospheres. This was accompanied by a reduction in the levels of NANOG, OCT4, and drug transporters. Exposure to RocA also induced cell death of the BCSCs as evaluated by DRAQ7 and cell viability assays. RocA treatment induced apoptosis with increased levels of cleaved caspase-3. Overall, we identified that RocA is effective in targeting BCSCs, and eIF4A is an actionable molecular target in both BCSCs and bulk tumor cells. Therefore, anti-eIF4A inhibitors could potentially be combined synergistically with existing chemo-, radio- and/or immunotherapies.

Novel and Alternative Targets Against Breast Cancer Stemness to Combat Chemoresistance.

Breast cancer stem cells (BCSCs) play a vital role in tumor progression and metastasis. They are heterogeneous and inherently radio- and chemoresistant. They have the ability to self-renew and differentiate into non-BCSCs. These determinants of BCSCs including the plasticity between the mesenchymal and epithelial phenotypes often leads to minimal residual disease (MRD), tumor relapse, and therapy failure. By studying the resistance mechanisms in BCSCs, a combinatorial therapy can be formulated to co-target BCSCs and bulk tumor cells. This review addresses breast cancer stemness and molecular underpinnings of how the cancer stemness can lead to pharmacological resistance. This might occur through rewiring of signaling pathways and modulated expression of various targets that support survival and self-renewal, clonogenicity, and multi-lineage differentiation into heterogeneous bulk tumor cells following chemotherapy. We explore emerging novel and alternative molecular targets against BC stemness and chemoresistance involving survival, drug efflux, metabolism, proliferation, cell migration, invasion, and metastasis. Strategic targeting of such vulnerabilities in BCSCs may overcome the chemoresistance and increase the longevity of the metastatic breast cancer patients.

The CXCR4-LASP1-eIF4F Axis Promotes Translation of Oncogenic Proteins in Triple-Negative Breast Cancer Cells.

Triple-negative breast cancer (TNBC) remains clinically challenging as effective targeted therapies are lacking. In addition, patient mortality mainly results from the metastasized lesions. CXCR4 has been identified to be one of the major chemokine receptors involved in breast cancer metastasis. Previously, our lab had identified LIM and SH3 Protein 1 (LASP1) to be a key mediator in CXCR4-driven invasion. To further investigate the role of LASP1 in this process, a proteomic screen was employed and identified a novel protein-protein interaction between LASP1 and components of eukaryotic initiation 4F complex (eIF4F). We hypothesized that activation of the CXCR4-LASP1-eIF4F axis may contribute to the preferential translation of oncogenic mRNAs leading to breast cancer progression and metastasis. To test this hypothesis, we first confirmed that the gene expression of CXCR4, LASP1, and eIF4A are upregulated in invasive breast cancer. Moreover, we demonstrate that LASP1 associated with eIF4A in a CXCL12-dependent manner via a proximity ligation assay. We then confirmed this finding, and the association of LASP1 with eIF4B via co-immunoprecipitation assays. Furthermore, we show that LASP1 can interact with eIF4A and eIF4B through a GST-pulldown approach. Activation of CXCR4 signaling increased the translation of oncoproteins downstream of eIF4A. Interestingly, genetic silencing of LASP1 interrupted the ability of eIF4A to translate oncogenic mRNAs into oncoproteins. This impaired ability of eIF4A was confirmed by a previously established 5'UTR luciferase reporter assay. Finally, lack of LASP1 sensitizes 231S cells to pharmacological inhibition of eIF4A by Rocaglamide A as evident through BIRC5 expression. Overall, our work identified the CXCR4-LASP1 axis to be a novel mediator in oncogenic protein translation. Thus, our axis of study represents a potential target for future TNBC therapies.

Novel Thienopyrimidine Derivative, RP-010, Induces ß-Catenin Fragmentation and Is Efficacious against Prostate Cancer Cells.

Thienopyrimidines containing a thiophene ring fused to pyrimidine are reported to have a wide-spectrum of anticancer efficacy in vitro. Here, we report for the first time that thieno[3,2-d]pyrimidine-based compounds, also known as the RP series, have efficacy in prostate cancer cells. The compound RP-010 was efficacious against both PC-3 and DU145 prostate cancer (PC) cells (IC50 < 1 µM). The cytotoxicity of RP-010 was significantly lower in non-PC, CHO, and CRL-1459 cell lines. RP-010 (0.5, 1, 2, and 4 µM) arrested prostate cancer cells in G2 phase of the cell cycle, and induced mitotic catastrophe and apoptosis in both PC cell lines. Mechanistic studies suggested that RP-010 (1 and 2 µM) affected the wingless-type MMTV (Wnt)/ß-catenin signaling pathway, in association with ß-catenin fragmentation, while also downregulating important proteins in the pathway, including LRP-6, DVL3, and c-Myc. Interestingly, RP-010 (1 and 2 µM) induced nuclear translocation of the negative feedback proteins, Naked 1 and Naked 2, in the Wnt pathway. In addition, RP-010 (0.5, 1, 2 and 4 µM) significantly decreased the migration of PC cells in vitro. Finally, RP-010 did not produce significant toxic effects in zebrafish at concentrations of up to 6 µM. In conclusion, RP-010 may be an efficacious and relatively nontoxic anticancer compound for prostate cancer. Future mechanistic and in vivo efficacy studies are needed to optimize the hit compound RP-010 for lead optimization and clinical use.

New Frontiers for the Cytoskeletal Protein LASP1.

In the recent two decades, LIM and SH3 protein 1 (LASP1) has been developed from a simple actin-binding structural protein to a tumor biomarker and subsequently to a complex, nuclear transcriptional regulator. Starting with a brief historical perspective, this review will mainly compare and contrast LASP1 and LASP2 from the angle of the newest data and importantly, examine their role in transcriptional regulation. We will summarize the current knowledge through pictorial models and tables including the roles of different microRNAs in the differential regulation of LASP1 levels and patient outcome rather than specify in detail all tumor entities. Finally, the novel functional roles of LASP1 in secretion of vesicles, expression of matrix metalloproteinases and transcriptional regulation as well as the activation of survival and proliferation pathways in different cancer types are described.

MDM2 Antagonists Counteract Drug-Induced DNA Damage.

Antagonists of MDM2-p53 interaction are emerging anti-cancer drugs utilized in clinical trials for malignancies that rarely mutate p53, including melanoma. We discovered that MDM2-p53 antagonists protect DNA from drug-induced damage in melanoma cells and patient-derived xenografts. Among the tested DNA damaging drugs were various inhibitors of Aurora and Polo-like mitotic kinases, as well as traditional chemotherapy. Mitotic kinase inhibition causes mitotic slippage, DNA re-replication, and polyploidy. Here we show that re-replication of the polyploid genome generates replicative stress which leads to DNA damage. MDM2-p53 antagonists relieve replicative stress via the p53-dependent activation of p21 which inhibits DNA replication. Loss of p21 promoted drug-induced DNA damage in melanoma cells and enhanced anti-tumor activity of therapy combining MDM2 antagonist with mitotic kinase inhibitor in mice. In summary, MDM2 antagonists may reduce DNA damaging effects of anti-cancer drugs if they are administered together, while targeting p21 can improve the efficacy of such combinations.

Connecting the Dots: Therapy-Induced Senescence and a Tumor-Suppressive Immune Microenvironment.

BACKGROUND: Tumor cell senescence is a common outcome of anticancer therapy. Here we investigated how therapy-induced senescence (TIS) affects tumor-infiltrating leukocytes (TILs) and the efficacy of immunotherapy in melanoma. METHODS: Tumor senescence was induced by AURKA or CDK4/6 inhibitors (AURKAi, CDK4/6i). Transcriptomes of six mouse tumors with differential response to AURKAi were analyzed by RNA sequencing, and TILs were characterized by flow cytometry. Chemokine RNA and protein expression were determined by quantitative real-time polymerase chain reaction and enzyme-linked immunosorbent assay. Therapeutic response was queried in immunodeficient mice, in mice with CCL5-deficient tumors, and in mice cotreated with CD137 agonist to activate TILs. CCL5 expression in reference to TIS and markers of TILs was studied in human melanoma tumors using patient-derived xenografts (n = 3 patients, n = 3 mice each), in AURKAi clinical trial samples (n = 3 patients, before/after therapy), and in The Cancer Genome Atlas (n = 278). All statistical tests were two-sided. RESULTS: AURKAi response was associated with induction of the immune transcriptome (P = 3.5 x 10-29) while resistance inversely correlated with TIL numbers (Spearman r = -0.87, P < .001). AURKAi and CDK4/6i promoted the recruitment of TILs by inducing CCL5 secretion in melanoma cells (P ≤ .005) in an NF-κB-dependent manner. Therapeutic response to AURKAi was impaired in immunodeficient compared with immunocompetent mice (0% vs 67% tumors regressed, P = .01) and in mice bearing CCL5-deficient vs control tumors (P = .61 vs P = .02); however, AURKAi response was greatly enhanced in mice also receiving T-cell-activating immunotherapy (P < .001). In human tumors, CCL5 expression was also induced by AURKAi (P ≤ .02) and CDK4/6i (P = .01) and was associated with increased immune marker expression (P = 1.40 x 10-93). CONCLUSIONS: Senescent melanoma cells secret CCL5, which promotes recruitment of TILs. Combining TIS with immunotherapy that enhances tumor cell killing by TILs is a promising novel approach to improve melanoma outcomes.