Repurposing FDA approved drugs with off-target autophagy inhibition such as chloroquine/hydroxychloroquine (CQ, HCQ) has produced modest anticancer activity in clinical trials, due in part, to a failure to define predictive biomarkers that enable the selection of patients that best respond to this treatment strategy. We identified a new role for REDD1 as a determinant of sensitivity to autophagy inhibition in renal cell carcinoma (RCC). RNA sequencing, qRT-PCR, immunoblotting, gene silencing, knockout and overexpression studies revealed that REDD1 expression is a key regulator of cell death stimulated by autophagy inhibitors. Comprehensive in vitro and in vivo studies were conducted to evaluate the selectivity, tolerability, and efficacy of the PIM kinase inhibitor TP-3654 and CQ in preclinical models of RCC. Markers of autophagy inhibition and cell death were evaluated in tumor specimens. Transcriptomic analyses identified REDD1 (DDIT4) as a highly induced gene in RCC cells treated with the PIM kinase inhibitor TP-3654. Focused studies confirmed that PIM1 inhibition was sufficient to induce REDD1 and stimulate autophagy through the AMPK cascade. DDIT4 knockout and overexpression studies established its mechanistic role as a regulator of sensitivity to autophagy inhibition. Inhibition of autophagy with CQ synergistically enhanced the in vitro and in vivo anticancer activity of TP-3654. Our findings identify REDD1 as a novel determinant of the sensitivity of RCC cells to autophagy inhibition and support further investigation of PIM kinase inhibition as a precision strategy to drive sensitivity to autophagy-targeted therapies through REDD1 upregulation.

Breakdown of every transmembrane protein trafficked to lysosomes requires proteolysis of their hydrophobic helical transmembrane domains. Combining lysosomal proteomics with functional genomic datasets, we identified lysosomal leucine aminopeptidase (LyLAP; formerly phospholipase B domain-containing 1) as the hydrolase most tightly associated with elevated endocytosis. Untargeted metabolomics and biochemical reconstitution demonstrated that LyLAP is a processive monoaminopeptidase with preference for amino-terminal leucine. This activity was necessary and sufficient for the breakdown of hydrophobic transmembrane domains. LyLAP was up-regulated in pancreatic ductal adenocarcinoma (PDA), which relies on macropinocytosis for nutrient uptake. In PDA cells, LyLAP ablation led to the buildup of undigested hydrophobic peptides, lysosomal membrane damage, and growth inhibition. Thus, LyLAP enables lysosomal degradation of membrane proteins and protects lysosomal integrity in highly endocytic cancer cells.

Statins, commonly used to lower cholesterol, are associated with improved prognosis in colorectal cancer (CRC), though their effectiveness varies. This study investigates the anti-cancer effects of atorvastatin in CRC using patient-derived organoids (PDOs) and PDO-derived xenograft (PDOX) models. Our findings reveal that atorvastatin induces mitochondrial dysfunction, leading to apoptosis in cancer cells. In response, cancer cells induce mitophagy to clear damaged mitochondria, enhancing survival and reducing statin efficacy. Analysis of a clinical cohort confirms mitophagy's role in diminishing statin effectiveness. Importantly, inhibiting mitophagy significantly enhances the anti-cancer effects of atorvastatin in CRC PDOs, xenograft models, and azoxymethane (AOM)-dextran sulfate sodium (DSS) mouse models. These findings identify mitophagy as a critical pro-survival mechanism in CRC during statin treatment, providing insights into the variable responses observed in epidemiological studies. Targeting this vulnerability through combination therapy can elicit potent therapeutic responses.

Neurofilament accumulation is associated with many neurodegenerative diseases, but it is the primary pathology in giant axonal neuropathy (GAN). This childhood-onset autosomal recessive disease is caused by loss-of-function mutations in gigaxonin, the E3 adaptor protein that enables neurofilament degradation. Using a combination of genetic and RNA interference approaches, we found that dorsal root ganglia from mice lacking gigaxonin have impaired autophagy and lysosomal degradation through 2 mechanisms. First, neurofilament accumulations interfere with the distribution of autophagic organelles, impairing their maturation and fusion with lysosomes. Second, the accumulations attract the chaperone 14-3-3, which is responsible for the proper localization of the key autophagy regulator transcription factor EB (TFEB). We propose that this dual disruption of autophagy contributes to the pathogenesis of other neurodegenerative diseases involving neurofilament accumulations.

The guanine exchange factor SOS1 plays a pivotal role in the positive feedback regulation of the KRAS signaling pathway. Recently, the regulation of KRAS-SOS1 interactions and KRAS downstream effector proteins has emerged as a key focus in the development of therapies targeting KRAS-driven cancers. However, the detailed dynamic mechanisms underlying SOS1-catalyzed GDP extraction and the impact of KRAS mutations remain largely unexplored. In this study, we unveil and describe in atomic detail the primary mechanisms by which SOS1 facilitates GDP extraction from KRAS oncogenes. For GDP-bound wild-type KRAS (KRAS-G12), four critical amino acids (Lys811, Glu812, Lys939, and Glu942) are identified as essential for the catalytic function of SOS1. Notably, the KRAS-G12D mutation (KRAS-D12) significantly accelerates the rate of GDP extraction. The molecular basis of this enhancement are attributed to hydrogen bonding interactions between the mutant residue Asp12 and a positively charged pocket in the intrinsically disordered region (residues 807-818), comprising Ser807, Trp809, Thr810, and Lys811. These findings provide novel insights into SOS1-KRAS interactions and offer a foundation for developing anti-cancer strategies aimed at disrupting these mechanisms.

Patient-derived organoids represent a novel platform to recapitulate the cancer cells in the patient tissue. While cancer heterogeneity has been extensively studied by a number of omics approaches, little is known about the spatiotemporal kinase activity dynamics. Here we applied a live imaging approach to organoids derived from 10 pancreatic ductal adenocarcinoma (PDAC) patients to comprehensively understand their heterogeneous growth potential and drug responses. By automated wide-area image acquisitions and analyses, the PDAC cells were non-selectively observed to evaluate their heterogeneous growth patterns. We monitored single-cell ERK and AMPK activities to relate cellular dynamics to molecular dynamics. Furthermore, we evaluated two anti-cancer drugs, a MEK inhibitor, PD0325901, and an autophagy inhibitor, hydroxychloroquine (HCQ), by our analysis platform. Our analyses revealed a phase-dependent regulation of PDAC organoid growth, where ERK activity is necessary for the early phase and AMPK activity is necessary for the late stage of organoid growth. Consistently, we found PD0325901 and HCQ target distinct organoid populations, revealing their combination is widely effective to the heterogeneous cancer cell population in a range of PDAC patient-derived organoid lines. Together, our live imaging quantitatively characterized the growth and drug sensitivity of human PDAC organoids at multiple levels: in single cells, single organoids, and individual patients. This study will pave the way for understanding the cancer heterogeneity and promote the development of new drugs that eradicate intractable cancer.

Neurofibromatosis type 1 (NF1) is a genetic disorder caused by mutation of the NF1 gene that is associated with various symptoms, including the formation of benign tumors, called neurofibromas, within nerves. Drug treatments are currently limited. The mitogen-activated protein kinase kinase (MEK) inhibitor selumetinib is used for a subset of plexiform neurofibromas (PNs) but is not always effective and can cause side effects. Therefore, there is a clear need to discover new drugs to target NF1-deficient tumor cells. Using a Drosophila cell model of NF1, we performed synthetic lethal screens to identify novel drug targets. We identified 54 gene candidates, which were validated with variable dose analysis as a secondary screen. Pathways associated with five candidates could be targeted using existing drugs. Among these, chloroquine (CQ) and bafilomycin A1, known to target the autophagy pathway, showed the greatest potential for selectively killing NF1-deficient Drosophila cells. When further investigating autophagy-related genes, we found that 14 out of 30 genes tested had a synthetic lethal interaction with NF1. These 14 genes are involved in multiple aspects of the autophagy pathway and can be targeted with additional drugs that mediate the autophagy pathway, although CQ was the most effective. The lethal effect of autophagy inhibitors was conserved in a panel of human NF1-deficient Schwann cell lines, highlighting their translational potential. The effect of CQ was also conserved in a Drosophila NF1 in vivo model and in a xenografted NF1-deficient tumor cell line grown in mice, with CQ treatment resulting in a more significant reduction in tumor growth than selumetinib treatment. Furthermore, combined treatment with CQ and selumetinib resulted in a further reduction in NF1-deficient cell viability. In conclusion, NF1-deficient cells are vulnerable to disruption of the autophagy pathway. This pathway represents a promising target for the treatment of NF1-associated tumors, and we identified CQ as a candidate drug for the treatment of NF1 tumors.

Our research group aimed for the optimization of pharmacologic ascorbate (Ph-Asc)-induced cancer cell death. To reduce the required time and resources needed for development, an in silico system biological approach, an already approved medication, and a mild bioactive compound were used in our previous studies. It was revealed that both Ph-Asc and resveratrol (RES) caused DSBs in the DNA, and chloroquine (CQ) treatment amplified the cytotoxic effect of both Ph-Asc and RES in an autophagy independent way. In the present study, we aimed at the further clarification of the cytotoxic mechanism of Ph-Asc, CQ, and RES by comparing their DNA damaging abilities, effects on the cells' bioenergetic status, ROS, and lipid ROS generation abilities with those of the three currently investigated compounds (menadione, RSL3, H2O2). It could be assessed that the induction of DSBs is certainly a common point of their mechanism of action; furthermore, the observed cancer cell death due to the investigated treatments are independent of the bioenergetic status. Contrary to other investigated compounds, the DNA damaging effect of CQ seemed to be ROS independent. Surprisingly, the well-known ferroptosis inducer RSL3 was unable to induce lipid peroxidation in the pancreas ductal adenocarcinoma (PDAC) Mia PaCa-2 cell line. At the same time, it induced DSBs in the DNA, and the RSL3-induced cell death could not be suspended by the well-known ferroptosis inhibitors. All these observations suggest the ferroptosis resistance of this cell line. The observed DNA damaging effect of RSL3 definitely creates a new perspective in anticancer research.

Mortality from colorectal cancer (CRC) is significant, and novel CRC therapies are needed. A pseudokinase MLKL typically executes necroptotic cell death, and MLKL inactivation protects cells from such death. However, we found unexpectedly that MLKL gene knockout enhanced CRC cell death caused by a protein synthesis inhibitor homoharringtonine used for chronic myeloid leukemia treatment. In an effort to explain this finding, we observed that MLKL gene knockout reduces the basal CRC cell autophagy and renders such autophagy critically dependent on the presence of VPS37A, a component of the ESCRT-I complex. We further found that the reason why homoharringtonine enhances CRC cell death caused by MLKL gene knockout is that homoharringtonine activates p38 MAP kinase and thereby prevents VPS37A from supporting autophagy in MLKL-deficient cells. We observed that the resulting inhibition of the basal autophagy in CRC cells triggers their parthanatos, a cell death type driven by poly(ADP-ribose) polymerase hyperactivation. Finally, we discovered that a pharmacological MLKL inhibitor necrosulfonamide strongly cooperates with homoharringtonine in suppressing CRC cell tumorigenicity in mice. Thus, while MLKL promotes cell death during necroptosis, MLKL supports the basal autophagy in CRC cells and thereby protects them from death. MLKL inactivation reduces such autophagy and renders the cells sensitive to autophagy inhibitors, such as homoharringtonine. Hence, MLKL inhibition creates a therapeutic vulnerability that could be utilized for CRC treatment.

Irreversible electroporation (IRE) is a local ablative treatment for patients with pancreatic cancer. During the IRE procedure, high-intensity electric pulses are released intratumorally to disrupt plasma membranes and induce cell death. Since the intensity of the pulsed electric field (PEF) can be decreased by the tumor microenvironment, some cancer cells are subjected to a sublethal PEF and may survive to cause tumor recurrence later. Autophagy activation induced by anticancer therapies is known to promote treatment resistance. In this study, we investigated whether autophagy is activated in residual cancer cells after IRE and assessed the roles it plays during tumor recurrence. Subcutaneous KPC-A548 or Panc02 murine pancreatic cancer cell line xenograft mouse models were established; once the tumors reached 7 mm in one dimension, the tumor-bearing mice were subjected to IRE. For in vitro sublethal PEF treatment, the pancreatic cancer cell suspension was in direct contact with the electrodes and pulsed at room temperature. We showed that autophagy was activated in surviving residual cells, as evidenced by increased expression of LC3 and p62. Suppression of autophagy with hydroxychloroquine (60 mg/kg, daily intraperitoneal injection) markedly increased the efficacy of IRE. We demonstrated that autophagy activation can be attributed to increased expression of high-mobility group box 1 (HMGB1); co-inhibition of two HMGB1 receptors, receptor for advanced glycosylation end products (RAGE) and Toll-like receptor 4 (TLR4), suppressed autophagy activation by upregulating the PI3K/AKT/p70 ribosomal S6 protein kinase (p70S6K) axis and sensitized pancreatic cancer cells to PEF. We prepared a polymeric micelle formulation (M-R/T) encapsulating inhibitors of both RAGE and TLR4. The combination of IRE and M-R/T (equivalent to RAGE inhibitor at 10.4 mg/kg and TLR4 inhibitor at 5.7 mg/kg, intravenous or intraperitoneal injection every other day) significantly promoted tumor apoptosis, suppressed cell cycle progression, and prolonged animal survival in pancreatic tumor models. This study suggests that disruption of HMGB1-mediated autophagy with nanomedicine is a promising strategy to enhance the response of pancreatic cancer to IRE.

Immunotherapy has become a crucial strategy in cancer treatment, but its effectiveness is often constrained. Most cancer immunotherapies focus on stimulating T-cell-mediated immunity by driving the cancer-immunity cycle, which includes tumor antigen release, antigen presentation, T cell activation, infiltration, and tumor cell killing. However, metabolism reprogramming in the tumor microenvironment (TME) supports the viability of cancer cells and inhibits the function of immune cells within this cycle, presenting clinical challenges. The distinct metabolic needs of tumor cells and immune cells require precise and selective metabolic interventions to maximize therapeutic outcomes while minimizing adverse effects. Recent advances in nanotherapeutics offer a promising approach to target tumor metabolism reprogramming and enhance the cancer-immunity cycle through tailored metabolic modulation. In this review, we explore cutting-edge nanomaterial strategies for modulating tumor metabolism to improve therapeutic outcomes. We review the design principles of nanoplatforms for immunometabolic modulation, key metabolic pathways and their regulation, recent advances in targeting these pathways for the cancer-immunity cycle enhancement, and future prospects for next-generation metabolic nanomodulators in cancer immunotherapy. We expect that emerging immunometabolic modulatory nanotechnology will establish a new frontier in cancer immunotherapy in the near future.

The resistance of pancreatic ductal adenocarcinoma (PDAC) to trametinib therapy limits its clinical use. However, the molecular mechanisms underlying trametinib resistance in PDAC remain unclear.

We aimed to illustrate the mechanisms of resistance to trametinib in PDAC and identify trametinib resistance-associated druggable targets, thus improving the treatment efficacy of trametinib-resistant PDAC.

We established patient-derived xenograft (PDX) models and primary cell lines to conduct functional experiments. We also applied single-cell RNA sequencing, Assay for Transposase-accessible Chromatin with sequencing and Cleavage Under Targets and Tagmentation sequencing to explore the relevant molecular mechanism.

We have identified a cancer cell subpopulation featured by hyperactivated viral mimicry response in trametinib-resistant PDXs. We have demonstrated that trametinib treatment of PDAC PDXs induces expression of transcription factor MAX dimerisation protein 1 (MXD1), which acts as a cofactor of histone methyltransferase mixed lineage leukaemia 1 to increased H3K4 trimethylation in transposable element (TE) loci, enhancing chromatin accessibility and thus the transcription of TEs. Mechanistically, enhanced transcription of TEs produces excessive double-stranded RNAs, leading to the activation of viral mimicry response and downstream oncogenic interferon-stimulated genes. Inhibiting MXD1 expression can recover the drug vulnerability of trametinib-resistant PDAC cells to trametinib.

Our study has discovered an important mechanism for trametinib resistance and identified MXD1 as a druggable target in treatment of trametinib-resistant PDAC.

The primary node molecules in the cell signaling network in cancer tissues are maladjusted and mutated in comparison to normal tissues, which promotes the occurrence and progression of cancer. Pancreatic cancer (PC) is a highly fatal cancer with increasing incidence and low five-year survival rates. Currently, there are several therapies that target cell signaling networks in PC. However, PC is a "cold tumor" with a unique immunosuppressive tumor microenvironment (poor effector T cell infiltration, low antigen specificity), and targeting a single gene or pathway is basically ineffective in clinical practice. Targeted matrix therapy, targeted metabolic therapy, targeted mutant gene therapy, immunosuppressive therapy, cancer vaccines, and other emerging therapies have shown great therapeutic potential, but results have been disappointing. Therefore, we summarize the identified and potential drug-resistant cell signaling networks aimed at overcoming barriers to existing PC therapies.

The Kirsten rat sarcoma viral oncogene homolog (KRAS) protein plays a key pathogenic role in oncogenesis, cancer progression, and metastasis. Numerous studies have explored the role of metabolic alterations in KRAS-driven cancers, providing a scientific rationale for targeting metabolism in cancer treatment. The development of KRAS-specific inhibitors has also garnered considerable attention, partly due to the challenge of acquired treatment resistance. Here, we review the metabolic reprogramming of glucose, glutamine, and lipids regulated by oncogenic KRAS, with an emphasis on recent insights into the relationship between changes in metabolic mechanisms driven by KRAS mutant and related advances in targeted therapy. We also focus on advances in KRAS inhibitor discovery and related treatment strategies in colorectal, pancreatic, and non-small cell lung cancer, including current clinical trials. Therefore, this review provides an overview of the current understanding of metabolic mechanisms associated with KRAS mutation and related therapeutic strategies, aiming to facilitate the understanding of current challenges in KRAS-driven cancer and to support the investigation of therapeutic strategies.

Malignant peripheral nerve sheath tumors (MPNSTs) are aggressive sarcomas and the primary cause of mortality in patients with neurofibromatosis type 1 (NF1). These malignancies develop within preexisting benign lesions called plexiform neurofibromas (PNs). PNs are solely driven by biallelic NF1 loss eliciting RAS pathway activation, and they respond favorably to MEK inhibitor therapy. MPNSTs harbor additional mutations and respond poorly to MEK inhibition. Our analysis of genetically engineered and orthotopic patient-derived xenograft MPNST models indicates that MEK inhibition has poor antitumor efficacy. By contrast, upstream inhibition of RAS through the protein-tyrosine phosphatase SHP2 reduced downstream signaling and suppressed NF1 MPNST growth, although resistance eventually emerged. To investigate possible mechanisms of acquired resistance, kinomic analyses of resistant tumors were performed, and data analysis identified enrichment of activated autophagy pathway protein kinases. Combining SHP2 inhibition with hydroxychloroquine (HQ) resulted in durable responses in NF1 MPNSTs in both genetic and orthotopic xenograft mouse models. Our studies could be rapidly translated into a clinical trial to evaluate SHP2 inhibition in conjunction with HQ as a unique treatment approach for NF1 MPNSTs.

Melanoma is a highly lethal form of skin cancer, and effective treatment remains a significant challenge. SPP86 is a novel potential therapeutic drug. Nonetheless, the specific influence of SPP86 on autophagy, particularly its mechanisms in the context of DNA damage and apoptosis in human melanoma cells, remains inadequately understood. Thus, this study aims to explore the effects of SPP86 on autophagy and to elucidate its association with cell proliferation, apoptosis, and DNA damage in melanoma cells.

This study assessed the anti-tumor effects of SPP86 on cell viability, colony formation, apoptosis, and DNA damage in two melanoma cell lines, A375 and A2058. Concurrently, the underlying mechanisms, including the PI3K/AKT signaling pathway and autophagy modulation, were also elucidated.

The study demonstrated that SPP86 exerts anti-tumor effects in melanoma cells through multiple mechanisms: it induces apoptosis, causes DNA damage, inhibits cell proliferation, and suppresses the PI3K/AKT signaling pathway. Importantly, the inhibition of autophagy appears to be a critical component of SPP86' s mode of action, with the modulation of autophagic processes influencing the cytotoxicity against melanoma cells.

These promising findings suggest that SPP86 is a potential drug candidate for the treatment of melanoma, warranting further research and development.

Pancreatic ductal adenocarcinoma (PDAC) poses a grim prognosis for patients. Recent multidisciplinary research efforts have provided critical insights into its genetics and tumor biology, creating the foundation for rational development of targeted and immune therapies. Here, we review the PDAC genomic landscape and the role of specific oncogenic events in tumor initiation and progression, as well as their contributions to shaping its tumor biology. We further summarize and synthesize breakthroughs in single-cell and metabolic profiling technologies that have illuminated the complex cellular composition and heterotypic interactions of the PDAC tumor microenvironment, with an emphasis on metabolic cross-talk across cancer and stromal cells that sustains anabolic growth and suppresses tumor immunity. These conceptual advances have generated novel immunotherapy regimens, particularly cancer vaccines, which are now in clinical testing. We also highlight the advent of KRAS targeted therapy, a milestone advance that has transformed treatment paradigms and offers a platform for combined immunotherapy and targeted strategies. This review provides a perspective summarizing current scientific and therapeutic challenges as well as practice-changing opportunities for the PDAC field at this major inflection point.

Background/Objectives: Preclinical studies have shown that the anti-malarial drug hydroxychloroquine (HCQ) improves the anti-cancer effects of various therapeutic agents by impairing autophagy. These findings are difficult to translate in vivo as reaching an effective HCQ concentration at the tumor site for extended times is challenging. Previously, we found that free HCQ in combination with gefitinib (Iressa®, ZD1839) significantly reduced tumor volume in immunocompromised mice bearing gefitinib-resistant JIMT-1 breast cancer xenografts. Here, we sought to evaluate whether a liposomal formulation of HCQ could effectively modulate autophagy in vivo and augment treatment outcomes in the same tumor model. Methods: We developed two liposomal formulations of HCQ: a pH-loaded formulation and a formulation based on copper complexation. The pharmacokinetics of each formulation was evaluated in CD1 mice following intravenous administration. An efficacy study was performed in immunocompromised mice bearing established JIMT-1tumors. Autophagy markers in tumor tissue harvested after four weeks of treatment were assessed by Western blot. Results: The liposomal formulations engendered ~850-fold increases in total drug exposure over time relative to the free drug. Both liposomal and free HCQ in combination with gefitinib provided comparable therapeutic benefits (p > 0.05). An analysis of JIMT-1 tumor tissue indicated that the liposomal HCQ and gefitinib combination augmented the inhibition of autophagy in vivo compared to the free HCQ and gefitinib combination as demonstrated by increased LC3-II and p62/SQSTM1 (p62) protein levels. Conclusions: The results suggest that liposomal HCQ has a greater potential to modulate autophagy in vivo compared to free HCQ; however, this did not translate to better therapeutic effects when used in combination with gefitinib to treat a gefitinib-resistant tumor model.

Proteolysis of hydrophobic helices is required for complete breakdown of every transmembrane protein trafficked to the lysosome and sustains high rates of endocytosis. However, the lysosomal mechanisms for degrading hydrophobic domains remain unknown. Combining lysosomal proteomics with functional genomic data mining, we identify Lysosomal Leucine Aminopeptidase (LyLAP; formerly Phospholipase B Domain-Containing 1) as the hydrolase most tightly associated with elevated endocytic activity. Untargeted metabolomics and biochemical reconstitution demonstrate that LyLAP is not a phospholipase, but a processive monoaminopeptidase with strong preference for N-terminal leucine - an activity necessary and sufficient for breakdown of hydrophobic transmembrane domains. LyLAP is upregulated in pancreatic ductal adenocarcinoma (PDA), which relies on macropinocytosis for nutrient uptake, and its ablation led to buildup of undigested hydrophobic peptides, which compromised lysosomal membrane integrity and inhibited PDA cell growth. Thus, LyLAP enables lysosomal degradation of membrane proteins, and may represent a vulnerability in highly endocytic cancer cells.

LyLAP degrades transmembrane proteins to sustain high endocytosis and lysosomal membrane stability in pancreatic cancer.

Pancreatic cancer (PC) is one of the most aggressive malignancies worldwide, and few effective therapeutics are available. Osthole (OST), a natural coumarin, has been proven to be a potential anticancer compound. In this study, we found that OST significantly inhibited PC growth both in vitro and in vivo. Notably, the anti-PC effect of OST is mediated by excessive autophagosome accumulation and consequent apoptosis in PC cells. Mechanistically, OST increased the number of autophagosomes via two pathways. First, OST induced autophagy initiation by suppressing the Akt/mTOR signaling pathway. Second, OST induced autophagic arrest by blocking autophagosome-lysosome fusion. Consistently, inhibition of autophagy initiation restored PC cell growth, whereas autophagic flux inhibitors exacerbated the antitumor effect of OST in PC cells, suggesting a cytotoxic role of OST-induced autophagosome accumulation. In addition, we found that OST upregulates the expression of activating transcription factor 3 (ATF3), resulting in inactivation of the Akt/mTOR signaling pathway and autophagy initiation in PC cells. ATF3 overexpression increased the activation of autophagy, and inhibition of ATF3 expression decreased autophagy. Taken together, our study provides new insights into the OST-induced growth inhibitory effect on PC cells, suggesting a promising potential therapeutic role of OST for PC treatment.

Present in all eukaryotic cells, the integrated stress response (ISR) is a highly coordinated signaling network that controls cellular behavior, metabolism, and survival in response to diverse stresses. The ISR is initiated when any 1 of 4 stress-sensing kinases (protein kinase R-like endoplasmic reticulum kinase [PERK], general control non-derepressible 2 [GCN2], double-stranded RNA-dependent protein kinase [PKR], heme-regulated eukaryotic translation initiation factor 2α kinase [HRI]) becomes activated to phosphorylate the protein translation initiation factor eukaryotic translation initiation factor 2α (eIF2α), shifting gene expression toward a comprehensive rewiring of cellular machinery to promote adaptation. Although the ISR has been shown to play an important role in the homeostasis of multiple tissues, evidence suggests that it is particularly crucial for the development and ongoing health of the pancreas. Among the most synthetically dynamic tissues in the body, the exocrine and endocrine pancreas relies heavily on the ISR to rapidly adjust cell function to meet the metabolic demands of the organism. The hardwiring of the ISR into normal pancreatic functions and adaptation to stress may explain why it is a commonly used pro-oncogenic and therapy-resistance mechanism in pancreatic ductal adenocarcinoma and pancreatic neuroendocrine tumors. Here, we review what is known about the key roles that the ISR plays in the development, homeostasis, and neoplasia of the pancreas.

Autophagy is an important catabolic process to maintain cellular homeostasis and antagonize cellular stresses. The initiation and activation are two of the most important aspects of the autophagic process. This review focuses on mechanisms underlying autophagy initiation and activation and signaling pathways regulating the activation of autophagy found in recent years. These findings include autophagy initiation by liquid-liquid phase separation (LLPS), autophagy initiation in the endoplasmic reticulum (ER) and Golgi apparatus, and the signaling pathways mediated by the ULK1 complex, the mTOR complex, the AMPK complex, and the PI3KC3 complex. Through the review, we attempt to present current research progress in autophagy regulation and forward our understanding of the regulatory mechanisms and signaling pathways of autophagy initiation and activation.

There has been growing interest in investigating anti-tumor drugs that not only kill cancer cells but also stimulate the immune system, among them, necroptosis is a classical immunogenic form of cell death. In our study, we discovered that by targeting RIP3, Jaspine B derivative C8 induces necroptosis and initiates cell death, and this effect can be reversed by knockout of RIP3. Furthermore, RIP3 initiates autophagy and binds to p62 to inhibit autophagic flux. Additionally, the autophagy process mediated by RIP3 activates the Nrf2 signaling pathway via the formation of the p62/Keap1 complex. Early autophagy inhibitors enhance necroptosis by impending the accumulation of p62 and restraining the activation of Nrf2, whereas late autophagy inhibitors partially prevent C8-induced necroptosis. Notably, the immunogenic form of cell death induced by C8 did not affect tumor immunity. Overall, C8 functions as a RIP3 activator to suppress the development of gastric cancer. Upon activation, RIP3 regulates p62-mediated autophagic flux and the Nrf2 signaling pathway through the RIP3/p62/Keap1 axis.

Cancer is a systemic heterogeneous disease involving complex molecular networks. Tumor formation involves an epithelial-mesenchymal transition (EMT), which promotes both metastasis and plasticity of cancer cells. Recent experiments have proposed that cancer cells can be transformed into adipocytes via a combination of drugs. However, the underlying mechanisms for how these drugs work, from a molecular network perspective, remain elusive. To reveal the mechanism of cancer-adipose conversion (CAC), this study adopts a systems biology approach by combing mathematical modeling and molecular experiments, based on underlying molecular regulatory networks. Four types of attractors are identified, corresponding to epithelial (E), mesenchymal (M), adipose (A) and partial/intermediate EMT (P) cell states on the CAC landscape. Landscape and transition path results illustrate that intermediate states play critical roles in the cancer to adipose transition. Through a landscape control approach, two new therapeutic strategies for drug combinations are identified, that promote CAC. These predictions are verified by molecular experiments in different cell lines. The combined computational and experimental approach provides a powerful tool to explore molecular mechanisms for cell fate transitions in cancer networks. The results reveal underlying mechanisms of intermediate cell states that govern the CAC, and identified new potential drug combinations to induce cancer adipogenesis.

Overcoming multiple barriers to deliver macromolecular drugs is an urgent challenge for glioma treatment. Herein, a strategy of protein corona-regulation synergizing with photoactivation based on T10 peptide-modified and indocyanine green (ICG)-loaded dendrigraft poly-L-lysines was proposed to augment prime editing therapy of glioma. First, the modified T10 peptide could escape the interference barrier of protein crown in blood via its specific binding with endogenous transferrin, thus crossing the blood-brain barrier (BBB) and achieving the targeting recognition of glioma cells. Next, the loaded ICG could weaken the tumor stromal barrier, decrease the cell membrane barrier and escape the lysosomal degradation/autophagy barrier via its photothermal and photodynamic effects. Subsequently, a therapeutic gene that could downregulate p-ERK1/2 for tumor growth inhibition and immunoregulation could be effectively delivered into the glioma cells. The glioma-targeted photo-gene combined immunotherapy effectively inhibit the glioma growth, especially co-dosing with the PD-1 antibody.

Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy that is often resistant to therapy. An immune suppressive tumor microenvironment (TME) and oncogenic mutations in KRAS have both been implicated as drivers of resistance to therapy. Mitogen-activated protein kinase (MAPK) inhibition has not yet shown clinical efficacy, likely because of rapid acquisition of tumor-intrinsic resistance. However, the unique PDAC TME may also be a driver of resistance. We found that long-term focal adhesion kinase (FAK) inhibitor treatment led to hyperactivation of the RAS/MAPK pathway in PDAC cells in mouse models and tissues from patients with PDAC. Concomitant inhibition of both FAK (with VS-4718) and rapidly accelerated fibrosarcoma and MAPK kinase (RAF-MEK) (with avutometinib) induced tumor growth inhibition and increased survival across multiple PDAC mouse models. In the TME, cancer-associated fibroblasts (CAFs) impaired the down-regulation of MYC by RAF-MEK inhibition in PDAC cells, resulting in resistance. By contrast, FAK inhibition reprogramed CAFs to suppress the production of FGF1, which can drive resistance to RAF-MEK inhibition. The addition of chemotherapy to combined FAK and RAF-MEK inhibition led to tumor regression, a decrease in liver metastasis, and improved survival in KRAS-driven PDAC mouse models. Combination of FAK and RAF-MEK inhibition alone improved antitumor immunity and priming of T cell responses in response to chemotherapy. These findings provided the rationale for an ongoing clinical trial evaluating the efficacy of avutometinib and defactinib in combination with gemcitabine and nab-paclitaxel in patients with PDAC and may suggest further paths for combined stromal and tumor-targeting therapies.

Outcomes in pancreatic ductal adenocarcinoma (PDAC) remain poor due to a variety of biological, clinical, and societal factors. However, in recent years, PDAC has seen 1) increased precision of initial evaluation, 2) increased emphasis on supportive care, 3) deeper understanding of the translation biology of PDAC, especially as pertains to genomic alterations, and 4) foundational combination chemotherapy clinical trials across all disease stages. These advances have led to a wide range of new approaches to drug therapy for PDAC. Currently available drugs are showing added benefit, both by resequencing them with each other and also with respect to other therapeutic modalities. Molecular strategies are being developed to predict response to known therapeutic agents and to identify others. Additionally, a wide range of new drugs for PDAC are under development, including drugs which inhibit critical molecular pathways, drugs which attempt to capitalize on homologous repair deficiencies, immunotherapeutic approaches, antimetabolic agents, and drugs which attack the extracellular matrix which supports PDAC growth. These new approaches offer the promise of improved survival for future PDAC patients.

Preclinical models of pancreatic cancer (PDAC) suggest a synergistic role for combined MEK and autophagy signaling inhibition, as well as MEK and CDK4/6 pathway targeting. Several case reports implicate clinical activity of the combination of either trametinib and hydroxychloroquine (HCQ) in patients with KRAS-mutant PDAC or trametinib with CDK4/6 inhibitors in patients with KRAS and CDKN2A/B alterations. However, prospective data from clinical trials is lacking. Here, we aim to provide clinical evidence regarding the use of these experimental regimens in the setting of dedicated precision oncology programs.

In this retrospective case series, PDAC patients who received either trametinib/HCQ (THCQ) or trametinib/palbociclib (TP) were retrospectively identified across 11 participating cancer centers in Germany.

Overall, 34 patients were identified. 19 patients received THCQ, and 15 received TP, respectively. In patients treated with THCQ, the median duration of treatment was 46 days, median progression-free survival (PFS) was 52 days and median overall survival (OS) was 68 days. In the THCQ subgroup, all patients evaluable for response (13/19) had progressive disease (PD) within 100 days. In the TP subgroup, the median duration of treatment was 60 days, median PFS was 56 days and median OS was 195 days. In the TP subgroup, 9/15 patients were evaluable for response, of which 1/9 showed a partial response (PR) while 8/9 had PD. One patient achieved a clinical benefit despite progression under TP.

THCQ and TP are not effective in patients with advanced PDAC harboring KRAS mutations or alterations in MAPK/CDKN2A/B.

Immune checkpoints are a set of inhibitory and stimulatory molecules/mechanisms that affect the activity of immune cells to maintain the existing balance between pro- and anti-inflammatory signaling pathways and avoid the progression of autoimmune disorders. Tumor cells can employ these checkpoints to evade immune system. The discovery and development of immune checkpoint inhibitors (ICIs) was thereby a milestone in the area of immuno-oncology. ICIs stimulate anti-tumor immune responses primarily by disrupting co-inhibitory signaling mechanisms and accelerate immune-mediated killing of tumor cells. Despite the beneficial effects of ICIs, they sometimes encounter some degrees of therapeutic resistance, and thereby do not effectively act against tumors. Among multiple combination therapies have been introduced to date, targeting autophagy, as a cellular degradative process to remove expired organelles and subcellular constituents, has represented with potential capacities to overcome ICI-related therapy resistance. It has experimentally been illuminated that autophagy induction blocks the immune checkpoint molecules when administered in conjugation with ICIs, suggesting that autophagy activation may restrict therapeutic challenges that ICIs have encountered with. However, the autophagy flux can also provoke the immune escape of tumors, which must be considered. Since the conventional FDA-approved ICIs have designed and developed to target programmed cell death receptor/ligand 1 (PD-1/PD-L1) as well as cytotoxic T lymphocyte-associated molecule 4 (CTLA-4) immune checkpoint molecules, we aim to review the effects of autophagy targeting in combination with anti-PD-1/PD-L1- and anti-CTLA-4-based ICIs on cancer therapeutic resistance and tumor immune evasion.

Pancreatic ductal adenocarcinoma (PDAC) presents significant challenges for targeted clinical interventions due to prevalent KRAS mutations, rendering PDAC resistant to RAF and MEK inhibitors (RAFi and MEKi). In addition, responses to targeted therapies vary between patients. Here, we explored the differential sensitivities of PDAC cell lines to RAFi and MEKi and developed an isogenic pair comprising the most sensitive and resistant PDAC cells. To simulate patient- or tumor-specific variations, we constructed cell-line-specific mechanistic models based on protein expression profiling and differential properties of KRAS mutants. These models predicted synergy between two RAFi with different conformation specificity (type I½ and type II RAFi) in inhibiting phospho-ERK (ppERK) and reducing PDAC cell viability. This synergy was experimentally validated across all four studied PDAC cell lines. Our findings underscore the need for combination approaches to inhibit the ERK pathway in PDAC.

Currently, a diagnosis with KRAS mutant pancreatic ductal adenocarcinoma (PDAC) means a death warrant, so finding efficient therapeutic options is a pressing issue. Here, we presented that pharmacologic ascorbate, chloroquine and resveratrol co-treatment exerted a synergistic cytotoxic effect on PDAC cell lines. The observed synergistic cytotoxicity was a general feature in all investigated cancer cell lines independent of the KRAS mutational status and seems to be independent of the autophagy inhibitory effect of chloroquine. Furthermore, it seems that apoptosis and necroptosis are also not likely to play any role in the cytotoxicity of chloroquine. Both pharmacologic ascorbate and resveratrol caused double-strand DNA breaks accompanied by cell cycle arrest. It seems resveratrol-induced cytotoxicity is independent of reactive oxygen species (ROS) generation and accompanied by a significant elevation of caspase-3/7 activity, while pharmacologic ascorbate-induced cytotoxicity shows strong ROS dependence but proved to be caspase-independent. Our results are particularly important since ascorbate and resveratrol are natural compounds without significant harmful effects on normal cells, and chloroquine is a known antimalarial drug that can easily be repurposed.

Macroautophagy/autophagy is primarily accountable for the degradation of damaged organelles and toxic macromolecules in the cells. Regarding the essential function of autophagy for preserving cellular homeostasis, changes in, or dysfunction of, autophagy flux can lead to disease development. In the current paper, the complicated function of autophagy in aging-associated pathologies and cancer is evaluated, highlighting the underlying molecular mechanisms that can affect longevity and disease pathogenesis. As a natural biological process, a reduction in autophagy is observed with aging, resulting in an accumulation of cell damage and the development of different diseases, including neurological disorders, cardiovascular diseases, and cancer. The MTOR, AMPK, and ATG proteins demonstrate changes during aging, and they are promising therapeutic targets. Insulin/IGF1, TOR, PKA, AKT/PKB, caloric restriction and mitochondrial respiration are vital for lifespan regulation and can modulate or have an interaction with autophagy. The specific types of autophagy, such as mitophagy that degrades mitochondria, can regulate aging by affecting these organelles and eliminating those mitochondria with genomic mutations. Autophagy and its specific types contribute to the regulation of carcinogenesis and they are able to dually enhance or decrease cancer progression. Cancer hallmarks, including proliferation, metastasis, therapy resistance and immune reactions, are tightly regulated by autophagy, supporting the conclusion that autophagy is a promising target in cancer therapy.

Mutational activation of KRAS occurs commonly in lung carcinogenesis and, with the recent U.S. Food and Drug Administration approval of covalent inhibitors of KRASG12C such as sotorasib or adagrasib, KRAS oncoproteins are important pharmacological targets in non-small cell lung cancer (NSCLC). However, not all KRASG12C-driven NSCLCs respond to these inhibitors, and the emergence of drug resistance in those patients who do respond can be rapid and pleiotropic. Hence, based on a backbone of covalent inhibition of KRASG12C, efforts are underway to develop effective combination therapies. Here, we report that the inhibition of KRASG12C signaling increases autophagy in KRASG12C-expressing lung cancer cells. Moreover, the combination of DCC-3116, a selective ULK1/2 inhibitor, plus sotorasib displays cooperative/synergistic suppression of human KRASG12C-driven lung cancer cell proliferation in vitro and superior tumor control in vivo. Additionally, in genetically engineered mouse models of KRASG12C-driven NSCLC, inhibition of either KRASG12C or ULK1/2 decreases tumor burden and increases mouse survival. Consequently, these data suggest that ULK1/2-mediated autophagy is a pharmacologically actionable cytoprotective stress response to inhibition of KRASG12C in lung cancer.