DrugMap: A quantitative pan-cancer analysis of cysteine ligandability.

Cysteine-focused chemical proteomic platforms have accelerated the clinical development of covalent inhibitors for a wide range of targets in cancer. However, how different oncogenic contexts influence cysteine targeting remains unknown. To address this question, we have developed "DrugMap," an atlas of cysteine ligandability compiled across 416 cancer cell lines. We unexpectedly find that cysteine ligandability varies across cancer cell lines, and we attribute this to differences in cellular redox states, protein conformational changes, and genetic mutations. Leveraging these findings, we identify actionable cysteines in NF-κB1 and SOX10 and develop corresponding covalent ligands that block the activity of these transcription factors. We demonstrate that the NF-κB1 probe blocks DNA binding, whereas the SOX10 ligand increases SOX10-SOX10 interactions and disrupts melanoma transcriptional signaling. Our findings reveal heterogeneity in cysteine ligandability across cancers, pinpoint cell-intrinsic features driving cysteine targeting, and illustrate the use of covalent probes to disrupt oncogenic transcription-factor activity.

Reductive stress in cancer: coming out of the shadows.

Redox imbalance is defined by disruption in oxidative and reductive pathways and has a central role in cancer initiation, development, and treatment. Although redox imbalance has traditionally been characterized by high levels of oxidative stress, emerging evidence suggests that an overly reductive environment is just as detrimental to cancer proliferation. Reductive stress is defined by heightened levels of antioxidants, including glutathione and elevated NADH, compared with oxidized NAD, which disrupts central biochemical pathways required for proliferation. With the advent of new technologies that measure and manipulate reductive stress, the sensors and drivers of this overlooked metabolic stress are beginning to be revealed. In certain genetically defined cancers, targeting reductive stress pathways may be an effective strategy. Redox-based pathways are gaining recognition as essential 'regulatory hubs,' and a broader understanding of reductive stress signaling promises not only to reveal new insights into metabolic homeostasis but also potentially to transform therapeutic options in cancer.

DrugMap: A quantitative pan-cancer analysis of cysteine ligandability.

Cysteine-focused chemical proteomic platforms have accelerated the clinical development of covalent inhibitors of a wide-range of targets in cancer. However, how different oncogenic contexts influence cysteine targeting remains unknown. To address this question, we have developed DrugMap , an atlas of cysteine ligandability compiled across 416 cancer cell lines. We unexpectedly find that cysteine ligandability varies across cancer cell lines, and we attribute this to differences in cellular redox states, protein conformational changes, and genetic mutations. Leveraging these findings, we identify actionable cysteines in NFκB1 and SOX10 and develop corresponding covalent ligands that block the activity of these transcription factors. We demonstrate that the NFκB1 probe blocks DNA binding, whereas the SOX10 ligand increases SOX10-SOX10 interactions and disrupts melanoma transcriptional signaling. Our findings reveal heterogeneity in cysteine ligandability across cancers, pinpoint cell-intrinsic features driving cysteine targeting, and illustrate the use of covalent probes to disrupt oncogenic transcription factor activity.

NRF2 activation induces NADH-reductive stress, providing a metabolic vulnerability in lung cancer.

Multiple cancers regulate oxidative stress by activating the transcription factor NRF2 through mutation of its negative regulator, KEAP1. NRF2 has been studied extensively in KEAP1-mutant cancers; however, the role of this pathway in cancers with wild-type KEAP1 remains poorly understood. To answer this question, we induced NRF2 via pharmacological inactivation of KEAP1 in a panel of 50+ non-small cell lung cancer cell lines. Unexpectedly, marked decreases in viability were observed in >13% of the cell lines-an effect that was rescued by NRF2 ablation. Genome-wide and targeted CRISPR screens revealed that NRF2 induces NADH-reductive stress, through the upregulation of the NAD+-consuming enzyme ALDH3A1. Leveraging these findings, we show that cells treated with KEAP1 inhibitors or those with endogenous KEAP1 mutations are selectively vulnerable to Complex I inhibition, which impairs NADH oxidation capacity and potentiates reductive stress. Thus, we identify reductive stress as a metabolic vulnerability in NRF2-activated lung cancers.

A T-cell independent universal cellular therapy strategy through antigen depletion.

Rationale: T cell therapeutic strategy using CD19-targeting chimeric antigen receptor (CAR) is a revolutionary, novel, and successful treatment for B-cell malignancies. However, the dependency on T-cell mediated cytotoxicity restricts CAR-T therapy as a patient-specific individualized therapy with severe side effects, such as cytokine release syndrome (CRS). Whether a non-T-cell based universal cellular therapy can substitute CAR-T therapy is largely unknown. Methods: Various artificial antigen-recognizing cells were prepared to determine whether non-T-cell-derived CD19-scFv bearing effector cells could cause target cell death. A universal strategy for CRS-free cellular therapeutics was proposed, utilizing artificial antigen-recognizing cells (AARC), which can be manufactured universally and routinely as "off-the-shelf" mesenchymal stromal cells (MSCs) or other types of non-autologous cells expressing anergic CARs. Results: We demonstrated that T-lymphocytic and non-lymphocytic cells could cause CD19 internalization and subsequent depletion when armed with a CD19-recognizing moiety. This CD19 antigen depletion could efficiently induce T-cell independent apoptosis in target cancer cells whose survival depends on CD19 expression, suggesting that CD19 antigen depletion constitutes a crucial tumor destroying mechanism for CD19-CAR-T, especially for its long-term efficacy. Conclusion: Our results uncovered an unrecognized CAR-T cytotoxicity and antigen loss mechanism and provided new insights into a shift from unique patient-specific autologous therapeutics to universal and standardized allogeneic treatment.

Deubiquitinating enzyme inhibitor alleviates cyclin A1-mediated proteasome inhibitor tolerance in mixed-lineage leukemia.

Drug resistance is a significant obstacle to effective cancer treatment. Drug resistance develops from initially reversible drug-tolerant cancer cells, which offer therapeutic opportunities to impede cancer relapse. The mechanisms of resistance to proteasome inhibitor (PI) therapy have been investigated intensively, however the ways by which drug-tolerant cancer cells orchestrate their adaptive responses to drug challenges remain largely unknown. Here, we demonstrated that cyclin A1 suppression elicited the development of transient PI tolerance in mixed-lineage leukemia (MLL) cells. This adaptive process involved reversible downregulation of cyclin A1, which promoted PI resistance through cell-cycle arrest. PI-tolerant MLL cells acquired cyclin A1 dependency, regulated directly by MLL protein. Loss of cyclin A1 function resulted in the emergence of drug tolerance, which was associated with patient relapse and reduced survival. Combination treatment with PI and deubiquitinating enzyme (DUB) inhibitors overcame this drug resistance by restoring cyclin A1 expression through chromatin crosstalk between histone H2B monoubiquitination and MLL-mediated histone H3 lysine 4 methylation. These results reveal the importance of cyclin A1-engaged cell-cycle regulation in PI resistance in MLL cells, and suggest that cell-cycle re-entry by DUB inhibitors may represent a promising epigenetic therapeutic strategy to prevent acquired drug resistance.

mTORC1-mediated amino acid signaling is critical for cell fate determination under transplant-induced stress.

Transplantation of in vitro-manipulated cells is widely used in hematology. While transplantation is well recognized to impose severe stress on transplanted cells, the nature of transplant-induced stress remains elusive. Here, we propose that the lack of amino acids in serum is the major cause of transplant-induced stress. Mechanistically, amino acid deficiency decreases protein synthesis and nutrient consummation. However, in cells with overactive AKT and ERK, mTORC1 is not inhibited and protein synthesis remains relatively high. This impaired signaling causes nutrient depletion, cell cycle block, and eventually autophagy and cell death, which can be inhibited by cycloheximide or mTORC1 inhibitors. Thus, mTORC1-mediated amino acid signaling is critical in cell fate determination under transplant-induced stress, and protein synthesis inhibition can improve transplantation efficiency.

Modulating proteasome inhibitor tolerance in multiple myeloma: an alternative strategy to reverse inevitable resistance.

BACKGROUND: Resistance to proteasome inhibitors (PIs) is a major obstacle to the successful treatment of multiple myeloma (MM). Many mechanisms have been proposed for PI resistance; however, our mechanistic understanding of how PI resistance is inevitably acquired and reversed remains incomplete. METHODS: MM patients after bortezomib relapse, MM cell lines and mouse models were used to generate matched resistant and reversed cells. RNA sequencing and bioinformatics analyses were employed to assess dysregulated epigenetic regulators. In vitro and in vivo procedures were used to characterise PI-tolerant cells and therapeutic efficacy. RESULTS: Upon PI treatment, MM cells enter a slow-cycling and reversible drug-tolerant state. This reversible phenotype is associated with epigenetic plasticity, which involves tolerance rather than persistence in patients with relapsed MM. Combination treatment with histone deacetylase inhibitors and high-dosage intermittent therapy, as opposed to sustained PI monotherapy, can be more effective in treating MM by preventing the emergence of PI-tolerant cells. The therapeutic basis is the reversal of dysregulated epigenetic regulators in MM patients. CONCLUSIONS: We propose an alternative non-mutational PI resistance mechanism that explains why PI relapse is inevitable and why patients regain sensitivity after a 'drug holiday'. Our study also suggests strategies for epigenetic elimination of drug-tolerant cells.

Integrative Analysis of Membrane Proteome and MicroRNA Reveals Novel Lung Cancer Metastasis Biomarkers.

Lung cancer is one of the most common human cancers both in incidence and mortality, with prognosis particularly poor in metastatic cases. Metastasis in lung cancer is a multifarious process driven by a complex regulatory landscape involving many mechanisms, genes, and proteins. Membrane proteins play a crucial role in the metastatic journey both inside tumor cells and the extra-cellular matrix and are a viable area of research focus with the potential to uncover biomarkers and drug targets. In this work we performed membrane proteome analysis of highly and poorly metastatic lung cells which integrated genomic, proteomic, and transcriptional data. A total of 1,762 membrane proteins were identified, and within this set, there were 163 proteins with significant changes between the two cell lines. We applied the Tied Diffusion through Interacting Events method to integrate the differentially expressed disease-related microRNAs and functionally dys-regulated membrane protein information to further explore the role of key membrane proteins and microRNAs in multi-omics context. Has-miR-137 was revealed as a key gene involved in the activity of membrane proteins by targeting MET and PXN, affecting membrane proteins through protein-protein interaction mechanism. Furthermore, we found that the membrane proteins CDH2, EGFR, ITGA3, ITGA5, ITGB1, and CALR may have significant effect on cancer prognosis and outcomes, which were further validated in vitro. Our study provides multi-omics-based network method of integrating microRNAs and membrane proteome information, and uncovers a differential molecular signatures of highly and poorly metastatic lung cancer cells; these molecules may serve as potential targets for giant-cell lung metastasis treatment and prognosis.

Restoring MLL reactivates latent tumor suppression-mediated vulnerability to proteasome inhibitors.

MLL undergoes multiple distinct chromosomal translocations to yield aggressive leukemia with dismal outcomes. Besides their well-established role in leukemogenesis, MLL fusions also possess latent tumor-suppressive activity, which can be exploited as effective cancer treatment strategies using pharmacological means such as proteasome inhibitors (PIs). Here, using MLL-rearranged xenografts and MLL leukemic cells as models, we show that wild-type MLL is indispensable for the latent tumor-suppressive activity of MLL fusions. MLL dysfunction, shown as loss of the chromatin accumulation and subsequent degradation of MLL, compromises the latent tumor suppression of MLL-AF4 and is instrumental for the acquired PI resistance. Mechanistically, MLL dysfunction is caused by chronic PI treatment-induced epigenetic reprogramming through the H2Bub-ASH2L-MLL axis and can be specifically restored by histone deacetylase (HDAC) inhibitors, which induce histone acetylation and recruits MLL on chromatin to promote cell cycle gene expression. Our findings not only demonstrate the mechanism underlying the inevitable acquisition of PI resistance in MLL leukemic cells, but also illustrate that preventing the emergence of PI-resistant cells constitutes a novel rationale for combination therapy with PIs and HDAC inhibitors in MLL leukemias.

Exosomes mediate intercellular transfer of non-autonomous tolerance to proteasome inhibitors in mixed-lineage leukemia.

Proteasome inhibitors significantly improve cancer outcomes, but their use is eventually followed by proteasome inhibitor resistance and relapse. Current understanding of proteasome inhibitor resistance is limited to cell-autonomous mechanisms; whether non-autonomous mechanisms can be implicated in the development of proteasome inhibitor resistance is unclear. Here, we show that proteasome inhibitor tolerance can be transmitted non-autonomously through exosome-mediated intercellular interactions. We revealed that reversible proteasome inhibitor resistance can be transmitted from cells under therapy stress to naïve sensitive cells through exosome-mediated cell cycle arrest and enhanced stemness in mixed-lineage leukemia cells. Integrated multi-omics analysis using the Tied Diffusion through Interacting Events algorithm identified several candidate exosomal proteins that may serve as predictors for proteasome inhibitor resistance and potential therapeutic targets for treating refractory mixed-lineage leukemia. Furthermore, inhibiting the secretion of exosomes is a promising strategy for reversing proteasome inhibitor resistance in vivo, which provides a novel proof of principle for the treatment of other refractory or relapsed cancers.

Arsenic sulfide induces RAG1-dependent DNA damage for cell killing by inhibiting NFATc3 in gastric cancer cells.

BACKGROUND: Arsenic sulfide was found to have potential anti-cancer activities, especially in gastric cancer. However, the underlying mechanism need to be further explored. This study was aimed to investigate the mechanism of arsenic compounds on gastric cancer. METHODS: Gastric cancer cell lines were infected with lentiviral vector carrying shNFATc3 and/or treated with arsenic sulfide. MTT assay were performed to assess cell growth. Flow cytometer assays were used to detect cell cycle and reactive oxygen species (ROS) level of gastric cancer cells. Western blot was carried out to detect nuclear factor of activated T-cells, cytoplasmic 3 (NFATc3), cell cycle markers, DNA damage pathway protein expression as well as other protein expression in gastric cancer cell lines. The expression of recombination activating gene 1 (RAG1) in gastric cancer cell lines was determined by RNA-sequencing analyses and Real-Time qPCR. The effect of NFATc3 on RAG1 were determined by CHIP-qPCR assay. The effect of arsenic sulfide on AGS cells was evaluated in vivo. RESULTS: We show that arsenic sulfide as well as knockdown of NFATc3 resulted in increased double-strand DNA damage in gastric cancer cells by increasing the expression of RAG1, an endonuclease essential for immunoglobulin V(D) J recombination. Overexpression of NFATc3 blocked the expression of RAG1 expression and DNA damage induced by arsenic sulfide. Arsenic sulfide induced cellular oxidative stress to redistribute NFATc3, thereby inhibiting its transcriptional function, which can be reversed by N-acetyl-L-cysteine (NAC). We show that NFATc3 targets the promoter of RAG1 for transcriptional inhibition. We further showed that NFATc3 upregulation and RAG1 downregulation significantly associated with poor prognosis in patients with gastric cancer. Our in vivo experiments further confirmed that arsenic sulfide exerted cytotoxic activity against gastric cancer cells through inhibiting NFATc3 to activate RAG1 pathway. CONCLUSION: These results demonstrate that arsenic sulfide targets NFATc3 to induce double strand DNA break (DSB) for cell killing through activating RAG1 expression. Our results link arsenic compound to the regulation of DNA damage control and RAG1 expression as a mechanism for its cytotoxic effect.

Cancer Cell Derived Small Extracellular Vesicles Contribute to Recipient Cell Metastasis Through Promoting HGF/c-Met Pathway.

Cancer progression is frequently caused by metastasis and leads to significantly increased mortality. Cell derived extracellular vesicles, including exosomes, in the microenvironment play key roles in cellular signal transduction, whereas their biological function in cancer metastasis and progression needs in-depth investigation. Here, we initially demonstrate that the small extracellular vesicles (sEVs) derived from highly metastatic lung cancer cells exhibited great capacity to promote the progression of recipient cells. Quantitative proteomics was employed to comprehensively decipher the proteome of cell derived sEVs and more than 1400 sEVs proteins were identified. Comparison analysis indicates that sEVs-HGF is a potential metastasis related protein and our verification data from clinical lung cancer plasma samples and in vivo experiments further confirmed the association. We found that sEVs-HGF could induce epithelial-mesenchymal transition and the coordination between HGF and c-Met was confirmed through corresponding target knockdown and kinase inhibition. Our data collectively demonstrate that cancer cell derived sEVs contribute to recipient cell metastasis through promoting HGF/c-Met pathway, which are potential targets for the prevention and treatment of cancer metastasis.

HERP Binds TBK1 To Activate Innate Immunity and Repress Virus Replication in Response to Endoplasmic Reticulum Stress.

Host innate immunity is crucial for cellular responses against viral infection sensed by distinct pattern recognition receptors and endoplasmic reticulum (ER) stress. Enterovirus 71 (EV71) is a causative agent of hand, foot, and mouth disease and neurological diseases. However, the exact mechanism underlying the link between ER stress induced by EV71 infection and host innate immunity is largely unknown. In this study, we demonstrated that EV71 infection induces the homocysteine-induced ER protein (HERP), a modulator of the ER stress response which is dependent on the participation of MAVS. Virus-induced HERP subsequently stimulates host innate immunity to repress viral replication by promoting type-I IFNs (IFN-α and IFN-ß) and type-III IFN (IFN-λ1) expression. Through interacting with TANK-binding kinase 1, HERP amplifies the MAVS signaling and facilitates the phosphorylation and nuclear translocation of IFN regulatory factor 3 and NF-κB to enhance the expression of IFNs, which leads to a broad inhibition of the replication of RNA viruses, including EV71, Sendai virus, influenza A virus, and vesicular stomatitis virus. Therefore, we demonstrated that HERP plays an important role in the regulation of host innate immunity in response to ER stress during the infection of RNA viruses. These findings provide new insights into the mechanism underlying the replication of RNA viruses and the production of IFNs, and also demonstrate a new role of HERP in the regulation of host innate immunity in response to viral infection.

HRS plays an important role for TLR7 signaling to orchestrate inflammation and innate immunity upon EV71 infection.

Enterovirus 71 (EV71) is an RNA virus that causes hand-foot-mouth disease (HFMD), and even fatal encephalitis in children. Although EV71 pathogenesis remains largely obscure, host immune responses may play important roles in the development of diseases. Recognition of pathogens mediated by Toll-like receptors (TLRs) induces host immune and inflammatory responses. Intracellular TLRs must traffic from the endoplasmic reticulum (ER) to the endolysosomal network from where they initiate complete signaling, leading to inflammatory response. This study reveals a novel mechanism underlying the regulation of TLR7 signaling during EV71 infection. Initially, we show that multiple cytokines are differentially expressed during viral infection and demonstrate that EV71 infection induces the production of proinflammatory cytokines through regulating TLR7-mediated p38 MAPK, and NF-κB signaling pathways. Further studies reveal that the expression of the endosome-associated protein hepatocyte growth factor-regulated tyrosine kinase substrate (HRS) is upregulated and highly correlated with the expression of TLR7 in EV71 infected patients, mice, and cultured cells. Virus-induced HRS subsequently enhances TLR7 complex formation in early- and late-endosome by interacting with TLR7 and TAB1. Moreover, HRS is involved in the regulation of the TLR7/NF-κB/p38 MAPK and the TLR7/NF-κB/IRF3 signaling pathways to induce proinflammatory cytokines and interferons, respectively, resulting in the orchestration of inflammatory and immune responses to the EV71 infection. Therefore, this study demonstrates that HRS acts as a key component of TLR7 signaling to orchestrate immune and inflammatory responses during EV71 infection, and provides new insights into the mechanisms underlying the regulation of host inflammation and innate immunity during EV71 infection.

Hepatitis B virus hijacks CTHRC1 to evade host immunity and maintain replication.

Hepatitis B virus (HBV) infection causes acute and chronic liver diseases, but is not directly cytopathic. Liver injury results from repeated attempts of the cellular immune response system to control the viral infection. Here, we investigate the roles of cellular factors and signaling pathways involved in the regulation of HBV replication to reveal the mechanism underlying HBV infection and pathogenesis. We show that collagen triple helix repeat containing 1 (CTHRC1) expression is elevated in HBV-infected patients and in HBV-transfected cells through epigenetic modification and transcriptional regulation. CTHRC1 facilitates HBV replication in cultured cells and BALB/c mice by activating the PKCα/ERK/JNK/c-Jun cascade to repress the IFN/JAK/STAT pathway. HBV-activated CTHRC1 downregulates the activity of type I interferon (IFN), the production of IFN-stimulated genes (ISGs), and the phosphorylation of signal transducer and activator of transcription 1/2 (STAT1/2), whereas it upregulates the phosphorylation and ubiquitination of type I IFN receptors (IFNARα/ß). Thus, our results show that HBV uses a novel mechanism to hijack cellular factors and signal cascades in order to evade host antiviral immunity and maintain persistent infection. We also demonstrate that CTHRC1 has a novel role in viral infection.