Chimeric antigen receptor (CAR) T cells have demonstrated promising efficacy, particularly in hematologic malignancies. One challenge regarding CAR T cells in solid tumors is the immunosuppressive ...tumor microenvironment (TME), characterized by high levels of multiple inhibitory factors, including transforming growth factor (TGF)-β. We report results from an in-human phase 1 trial of castration-resistant, prostate cancer-directed CAR T cells armored with a dominant-negative TGF-β receptor (NCT03089203). Primary endpoints were safety and feasibility, while secondary objectives included assessment of CAR T cell distribution, bioactivity and disease response. All prespecified endpoints were met. Eighteen patients enrolled, and 13 subjects received therapy across four dose levels. Five of the 13 patients developed grade ≥2 cytokine release syndrome (CRS), including one patient who experienced a marked clonal CAR T cell expansion, >98% reduction in prostate-specific antigen (PSA) and death following grade 4 CRS with concurrent sepsis. Acute increases in inflammatory cytokines correlated with manageable high-grade CRS events. Three additional patients achieved a PSA reduction of ≥30%, with CAR T cell failure accompanied by upregulation of multiple TME-localized inhibitory molecules following adoptive cell transfer. CAR T cell kinetics revealed expansion in blood and tumor trafficking. Thus, clinical application of TGF-β-resistant CAR T cells is feasible and generally safe. Future studies should use superior multipronged approaches against the TME to improve outcomes.
Clinical trials repurposing lysosomotropic chloroquine (CQ) derivatives as autophagy inhibitors in cancer demonstrate encouraging results, but the underlying mechanism of action remains unknown. ...Here, we report a novel dimeric CQ (DC661) capable of deacidifying the lysosome and inhibiting autophagy significantly better than hydroxychloroquine (HCQ). Using an
photoaffinity pulldown strategy, we identified palmitoyl-protein thioesterase 1 (PPT1) as a molecular target shared across monomeric and dimeric CQ derivatives. HCQ and Lys05 also bound to and inhibited PPT1 activity, but only DC661 maintained activity in acidic media. Knockout of PPT1 in cancer cells using CRISPR/Cas9 editing abrogates autophagy modulation and cytotoxicity of CQ derivatives, and results in significant impairment of tumor growth similar to that observed with DC661. Elevated expression of PPT1 in tumors correlates with poor survival in patients in a variety of cancers. Thus, PPT1 represents a new target in cancer that can be inhibited with CQ derivatives. SIGNIFICANCE: This study identifies PPT1 as the previously unknown lysosomal molecular target of monomeric and dimeric CQ derivatives. Genetic suppression of PPT1 impairs tumor growth, and PPT1 levels are elevated in cancer and associated with poor survival. These findings provide a strong rationale for targeting PPT1 in cancer.
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Chimeric antigen receptor (CAR) T cells have not induced meaningful clinical responses in solid tumors. Loss of T cell stemness, poor expansion capacity, and exhaustion during prolonged tumor antigen ...exposure are major causes of CAR T cell therapeutic resistance. Single-cell RNA-sequencing analysis of CAR T cells from a first-in-human trial in metastatic prostate cancer identified two independently validated cell states associated with antitumor potency or lack of efficacy. Low expression of
, encoding the BLIMP1 transcription factor, defined highly potent
encoding T cell factor 1 (TCF1)-expressing CD8
CAR T cells, whereas enrichment of
encoding T cell immunoglobulin and mucin-domain containing-3 (TIM-3)-expressing CD8
T cells with elevated
was associated with poor outcomes.
knockout promoted
-dependent CAR T cell stemness and proliferation, resulting in marginally enhanced leukemia control in mice. However, in the setting of
deficiency, a negative epigenetic feedback program of nuclear factor of activated T cells (NFAT)-driven T cell dysfunction was identified. This program was characterized by compensatory up-regulation of
and other genes encoding exhaustion-related transcription factors that hampered T cell effector function in solid tumors. Dual knockout of
and
skewed CAR T cell phenotypes away from TIM-3
CD8
and toward TCF1
CD8
to counter exhaustion of tumor-infiltrating CAR T cells and improve antitumor responses, effects that were not achieved with
and
single knockout alone. These data underscore dual targeting of
and
as a promising approach to advance adoptive cell immuno-oncotherapy.
Lysosomes serve dual roles in cancer metabolism, executing catabolic programs (i.e., autophagy and macropinocytosis) while promoting mTORC1-dependent anabolism. Antimalarial compounds such as ...chloroquine or quinacrine have been used as lysosomal inhibitors, but fail to inhibit mTOR signaling. Further, the molecular target of these agents has not been identified. We report a screen of novel dimeric antimalarials that identifies dimeric quinacrines (DQ) as potent anticancer compounds, which concurrently inhibit mTOR and autophagy. Central nitrogen methylation of the DQ linker enhances lysosomal localization and potency. An
photoaffinity pulldown identified palmitoyl-protein thioesterase 1 (PPT1) as the molecular target of DQ661. PPT1 inhibition concurrently impairs mTOR and lysosomal catabolism through the rapid accumulation of palmitoylated proteins. DQ661 inhibits the
tumor growth of melanoma, pancreatic cancer, and colorectal cancer mouse models and can be safely combined with chemotherapy. Thus, lysosome-directed PPT1 inhibitors represent a new approach to concurrently targeting mTORC1 and lysosomal catabolism in cancer.
This study identifies chemical features of dimeric compounds that increase their lysosomal specificity, and a new molecular target for these compounds, reclassifying these compounds as targeted therapies. Targeting PPT1 blocks mTOR signaling in a manner distinct from catalytic inhibitors, while concurrently inhibiting autophagy, thereby providing a new strategy for cancer therapy.
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Many sarcomas and leukemias carry nonrandom chromosomal translocations encoding tumor-specific mutant fusion transcription factors that are essential to their molecular pathogenesis. Ewing's sarcoma ...family tumors (ESFTs) contain a characteristic t(11;22) translocation leading to expression of the oncogenic fusion protein EWS-FLI1. EWS-FLI1 is a disordered protein that precludes standard structure-based small-molecule inhibitor design. EWS-FLI1 binding to RNA helicase A (RHA) is important for its oncogenic function. We therefore used surface plasmon resonance screening to identify compounds that bind EWS-FLI1 and might block its interaction with RHA. YK-4-279, a derivative of the lead compound from the screen, blocks RHA binding to EWS-FLI1, induces apoptosis in ESFT cells and reduces the growth of ESFT orthotopic xenografts. These findings provide proof of principle that inhibiting the interaction of mutant cancer-specific transcription factors with the normal cellular binding partners required for their oncogenic activity provides a promising strategy for the development of uniquely effective, tumor-specific anticancer agents.
Oncogenic fusion proteins, such as EWS-FLI1, are excellent therapeutic targets as they are only located within the tumor. However, there are currently no agents targeted toward transcription factors, ...which are often considered to be 'undruggable.' A considerable body of evidence is accruing that refutes this claim based upon the intrinsic disorder of transcription factors. Our previous studies show that RNA Helicase A (RHA) enhances the oncogenesis of EWS-FLI1, a putative intrinsically disordered protein. Interruption of this protein-protein complex by small molecule inhibitors validates this interaction as a unique therapeutic target. Single enantiomer activity from a chiral compound has been recognized as strong evidence for specificity in a small molecule-protein interaction. Our compound, YK-4-279, has a chiral center and can be separated into two enantiomers by chiral HPLC. We show that there is a significant difference in activity between the two enantiomers. (S)-YK-4-279 is able to disrupt binding between EWS-FLI1 and RHA in an immunoprecipitation assay and blocks the transcriptional activity of EWS-FLI1, while (R)-YK-4-279 cannot. Enantiospecific effects are also established in cytotoxicity assays and caspase assays, where up to a log-fold difference is seen between (S)-YK-4-279 and the racemic YK-4-279. Our findings indicate that only one enantiomer of our small molecule is able to specifically target a protein-protein interaction. This work is significant for its identification of a single enantiomer effect upon a protein interaction suggesting that small molecule targeting of intrinsically disordered proteins can be specific. Furthermore, proving YK-4-279 has only one functional enantiomer will be helpful in moving this compound towards clinical trials.
Aberrant activation of S6 kinase 1 (S6K1) is found in many diseases, including diabetes, aging, and cancer. We developed ATP competitive organometallic kinase inhibitors, EM5 and FL772, which are ...inspired by the structure of the pan-kinase inhibitor staurosporine, to specifically inhibit S6K1 using a strategy previously used to target other kinases. Biochemical data demonstrate that EM5 and FL772 inhibit the kinase with IC50 value in the low nanomolar range at 100 μM ATP and that the more potent FL772 compound has a greater than 100-fold specificity over S6K2. The crystal structures of S6K1 bound to staurosporine, EM5, and FL772 reveal that the EM5 and FL772 inhibitors bind in the ATP binding pocket and make S6K1-specific contacts, resulting in changes to the p-loop, αC helix, and αD helix when compared to the staurosporine-bound structure. Cellular data reveal that FL772 is able to inhibit S6K phosphorylation in yeast cells. Together, these studies demonstrate that potent, selective, and cell permeable S6K1 inhibitors can be prepared and provide a scaffold for future development of S6K inhibitors with possible therapeutic applications.
Ewing tumor is driven by the oncogenic EWS-FLI1 fusion protein that functions as an aberrant transcription factor. The identification of EWS-FLI1 protein partners is essential to enhance its ...vulnerability as a therapeutic target. We utilized phage display library screening against recombinant EWS-FLI1 protein. We identified 27 unique Ewing sarcoma binding peptides. The cytotoxicity evaluation of these peptides with in EWS-FLI1 containing cell lines yielded one potent peptide called ESAP1 (TMRGKKKRTRAN). ESAP1 binds EWS-FLI1 with 0.202 micromolar affinity as measured in surface plasmon resonance. The minimal interaction region of ESAP1 is characterized and found that the lysine residues are critical for cellular cytotoxicity. ESAP1 reduces the transcriptional activity of EWS-FLI1 as well as disrupts cell cycle kinetics in Ewing Tumor cells. These findings provide both a novel experimental probe and a potential therapeutic scaffold for Ewing Tumor.
Transcription factors have long been deemed 'undruggable' targets for therapeutics. Enhanced recognition of protein biochemistry as well as the need to have more targeted approaches to treat cancer ...has rendered transcription factors approachable for therapeutic development. Since transcription factors lack enzymatic domains, the specific targeting of these proteins has unique challenges. One challenge is the hydrophobic microenvironment that affects small molecules gaining access to block protein interactions. The most attractive transcription factors to target are those formed from tumor specific chromosomal translocations that are validated oncogenic driver proteins. EWS-FLI1 is a fusion protein that results from the pathognomonic translocation of Ewing sarcoma (ES). Our past work created the small molecule YK-4-279 that blocks EWS-FLI1 from interacting with RNA Helicase A (RHA). To fulfill long-standing promise in the field by creating a clinically useful drug, steps are required to allow for in vivo administration. These investigations identify the need for continuous presence of the small molecule protein-protein inhibitor for a period of days. We describe the pharmacokinetics of YK-4-279 and its individual enantiomers. In vivo studies confirm prior in vitro experiments showing (S)-YK-4-279 as the EWS-FLI1 specific enantiomer demonstrating both induction of apoptosis and reduction of EWS-FLI1 regulated caveolin-1 protein. We have created the first rat xenograft model of ES, treated with (S)-YK-4-279 dosing based upon PK modeling leading to a sustained complete response in 2 of 6 ES tumors. Combining laboratory studies, pharmacokinetic measurements, and modeling has allowed us to create a paradigm that can be optimized for in vivo systems using both in vitro data and pharmacokinetic simulations. Thus, (S)-YK-4-279 as a small molecule drug is ready for continued development towards a first-in-human, first-in-class, clinical trial.
Ewing Sarcoma (ES) is associated with a balanced chromosomal translocation that in most cases leads to the expression of the oncogenic fusion protein and transcription factor EWS-FLI1. EWS-FLI1 has ...been shown to be crucial for ES cell survival and tumor growth. However, its regulation is still enigmatic. To date, no functionally significant post-translational modifications of EWS-FLI1 have been shown. Since ES are sensitive to histone deacetylase inhibitors (HDI), and these inhibitors are advancing in clinical trials, we sought to identify if EWS-FLI1 is directly acetylated. We convincingly show acetylation of the C-terminal FLI1 (FLI1-CTD) domain, which is the DNA binding domain of EWS-FLI1. In vitro acetylation studies showed that acetylated FLI1-CTD has higher DNA binding activity than the non-acetylated protein. Over-expression of PCAF or treatment with HDI increased the transcriptional activity of EWS-FLI1, when co-expressed in Cos7 cells. However, our data that evaluates the acetylation of full-length EWS-FLI1 in ES cells remains unclear, despite creating acetylation specific antibodies to four potential acetylation sites. We conclude that EWS-FLI1 may either gain access to chromatin as a result of histone acetylation or undergo regulation by direct acetylation. These data should be considered when patients are treated with HDAC inhibitors. Further investigation of this phenomenon will reveal if this potential acetylation has an impact on tumor response.
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