FASN inhibition (FASNi) has been promising in mutant KRAS lung cancer (KMLC) preclinical models and patients (NCT03808558). Our lab described that KMLC cells treated with FASNi undergo ferroptosis, a form of iron- and reactive oxygen species (ROS)-dependent cell death characterized by lipid peroxidation (LPO). Indeed, we found that FASN provides new fatty acids in order to repair LPO caused under the high ROS in KMLC. To gain insights into the potential mechanisms of acquired resistance to FASNi-induced ferroptosis and to establish the rationale for future clinical trials, we developed an in vitro model of FASNi-resistance using human KMLC cell lines. A CRISPR/Cas9 screening and functional experiments showed that the histone lysine demethylase 6B (KDM6B) mediates the resistance of KMLC to FASNi in vitro and in vivo. KDM6B specifically demethylates H3K27me2/3, thereby activating gene expression. Therefore, we performed RNA-seq on isogenic pairs of FASNi-resistant and sensitive LC cells treated either with FASNi or the KDM6 inhibitor GSK-J4. We found that only GSK-J4 downregulates lipid metabolism genes in resistant cells. Using a pharmacogenomic approach and lipidomics we found that the gene signature induced by GSK-J4 significantly overlaps with that of known ferroptosis inducers and that FASNi resistant cells undergo LPO upon KDM6 inhibition. These data suggest that KDM6B transcriptionally regulates lipid metabolism and ferroptosis in KMLC resistant to FASNi. To get additional insights, Chromatin immunoprecipitation-sequencing (ChIP-seq) experiments are ongoing. Building upon these data, we expect to determine unprecedented connections among de novo lipid synthesis, epigenetics, drug resistance and ferroptosis.

Immunotherapy has raised unprecedent expectations in the treatment of virtually every cancer. However, despite some remarkable successes, resistance remains an unsurmountable hurdle.  Using TCB and CARs directed against HER2, we demonstrate that disruption in interferon-gamma signaling in cancer cells is a mechanism of intrinsic resistance to killing by fully active, correctly engaged, T lymphocytes. Importantly, we have identified that the kinase JAK2, which transduces the signal initiated by interferon-gamma, is the component preferably disrupted to acquired resistance in all resistant models developed in vitro and in vivo. These results unveil a novel mechanism of resistance to T-cell based therapies, and imply the potential use of JAK2 and IFNgamma response as a surrogate biomarker of response to immunotherapies. In addition, they open the avenue for the screening for therapies that can overcome deficient IFNgamma response or restore JAK2 levels, which are promising potential candidates to increase the benefits of immunotherapies. 

Enteric glial cells (EGC) have recently been shown to impact tumor development. Here, we investigate the impact of EGC on cancer stem cell (CSC) chemoresistance. EGC promoted growth of CSC-derived tumors in the presence of 5-FU in vivo and in vitro. EGC-conditioned medium (CM) reduced 5-FU-induced apoptosis in CSC. ATM mRNA was significantly enriched in 5-FU-treated CSC grown with EGC vs. alone. EGC pro-chemoresistance effects were reduced in CSC derived from cell lines knocked-down for ATM. Inhibition of ATM activity using KU-55933, or upstream MRN activity using mirin abolished EGC-induced CSC resistance to 5-FU. Mass spectrometric analyses identified Follistatin Like 3 (FSTL3) was highly enriched in the CM of 5-FU-treated EGC. Treatment with recombinant FSTL3 increased tumor formation and reduced apoptosis in CSC in the presence of 5-FU. Altogether our data show that EGC stimulate CSC chemoresistance using the MRN/ATM pathway, potentially through the release of FSTL3.

In triple negative breast cancer (TNBC), resistance to chemotherapy is a major clinical issue leading to the worst prognosis among all breast cancer subtypes. Using established TNBC models of acquired resistance to chemotherapy, we have evidenced the role of the H3K27me3 repressive histone mark as a lock for the adaptation of cancer cells to therapeutic stress. In order to propose novel therapies against recurrence, we are challenging the H3K27me3 balance and deciphering both the contributions of H3K27 demethylases (KDMs) and methyltransferases (KMTs) to chemoresistance in TNBC. By investigating their functions with loss-of-function models, we are understanding how these enzymes modulate the response to chemotherapy at different timepoints. Furthermore, by capturing the dynamics of their binding patterns and determining the repertoire of their protein interactions overtime in the context of chemotherapy, we will provide a more comprehensive view of H3K27 KDMs and KMTs roles for the emergence of resistance in TNBC.

Medulloblastoma (MB) is the deadliest brain tumor of childhood, intrinsically characterized by fast growth, high invasiveness, and resistance to treatments. To deepen the molecular basis of MB aggressiveness and recurrence, we established an in vitro model of MB resistance to chemotherapy. Preliminary data on our model show that resistant cells induce the activation of the two main regulator of pentose phosphate pathway TKT and G6PD via the activation of Nrf2. Moreover, resistant cells show an increase in HIF-1α signaling together with an augmented expression of glycolytic enzymes HK1, PFKB-3, PDK1 and LDHA and increased glycolytic capacity.    

Interestingly, enrichment analysis on label free mass spectrometry data reveal that the most significant terms deriving from the upregulated proteins, that characterized resistant cells, were related to metabolic processes such as “carbon metabolism”, “fatty acid beta oxidation”, “tricarboxylic acid cycle” and “carboxylic acid catabolic processes”. 

Consistently with recent studies that highlight the relevance of metabolic plasticity of cancer cells in chemotherapy adaptation, our data suggest a metabolic uncoupling in which MB resistant cells enhance glycolytic rate for metabolite production to counteract chemotherapy insults, while improving the oxidative metabolism to sustain energy needs.

A major challenge in the treatment of kinase driven tumors is the inevitable emergence of resistance and the limited efficacy of subsequent treatments including immunotherapy. We therefore systematically studied the mechanisms enabling cell persistence despite targeted kinase inhibition across kinase driven tumor models of different entities. Time-series RNA-seq displayed expression of an interferon gene signature and a senescence-associated inflammatory response. This included upregulation of the antigen presenting machinery and of nucleic acid receptors MDA-5, TLR3 and RIG-I. Mechanistically, chemical and genomic perturbations indicate that this response is dependent on IRF1 upregulation driven by MAPK inhibition. Humanized xenografts and syngeneic mouse models of EGFR-driven lung cancer showed that addition of RIG-I agonist IVT4 to EGFR inhibition increased tumor shrinkage in an NK- and CD8 cell dependent manner. Our findings thus provide a novel treatment strategy exploiting the adaptation mechanisms to targeted kinase inhibition through rational combination with an immunotherapeutic agent.

Resistance to radiotherapy is a major obstacle in cancer therapy. Both cancer-associated fibroblasts (CAFs) within the tumor microenvironment (TME) and Notch signaling are implicated in radioresistance, but their potential interrelationship is unclear. Here, we report that samples obtained from luminal breast cancer patient tumors irradiated in a neoadjuvant setting express higher levels of the Notch ligand Dll1 and a greater number of αSMA- and FAP-expressing activated CAFs. Single cell transcriptomic profiles further reveal enrichment of a Dll1-dependent αSMA⁺ myofibroblastic CAF subpopulation. Genetic and pharmacological blocking of Dll1 mediated Notch signaling decreased the number of Dll1⁺ cancer stem cells (CSCs) and CAFs, and increased tumor cell radiosensitivity, thereby highlighting essential crosstalk between Dll1⁺ tumor cells and CAFs in driving radioresistance. In support, we found that CAFs secrete Wnt ligands in a Dll1-dependent fashion, thereby promoting Wnt/β-catenin dependent signaling increases in Dll1⁺ CSC function. Notably, the porcupine inhibitor, LGK-974, which inhibits Wnt ligand secretion, as well as pharmacological blocking with anti-IL6 which we found to recruit CAFs to the TME and anti-DLL1 antibodies can all suppress CAF-dependent enhancement of Dll1⁺ CSC function and metastasis in radioresistant tumors. Together, these findings uncover crosstalk between Dll1⁺ cancer cells and CAFs that promotes metastases and radioresistance, which could be exploited in future therapeutic applications in radioresistant breast cancer patients.

Non-small cell lung cancer (NSCLC) is the leading cause of death worldwide. Genetic alterations of EGFR lead to a constitutive activation of EGFR tyrosine kinase activity in lung adenocarcinoma. The discovery of EGFR tyrosine kinase inhibitor (TKI) such as gefitinib or erlotinib exhibits a great success against activating mutation. However, acquired resistance emerges after a period of treatment largely attributed to a secondary EGFR T790M mutation. Although T790M-specific third generation TKI, osimertinib (AZD9291) can overcome the acquired resistance, its effect is hindered by a newly developed acquired resistance. Therefore, exploring resistance mechanisms is critical for lung adenocarcinoma harboring EGFR activation mutation.

NSCLC cells with acquired resistance to the first and third generation of EGFR-TKIs were generated not exhibiting additional EGFR mutation (HCC827/IR and H1975/AR cells, respectively). Epithelial-mesenchymal transition and cancer stem cell feature were revealed. To further explore whether common mediator participates in this event, RNA-seq is performed and identified the upregulation of AKT3 but not AKT1 and AKT2. Immunoprecipitation of AKT3 demonstrates its contribution to AKT activation (p-AKT). 

Gefitinib and AZD9291 further increased AKT3 protein level in both resistant cells. Elevated AKT3 protein was not affected by proteasome inhibitor MG132, indicating that proteasomal degradation was reduced in this event. Slower degradation in the presence of cycloheximide was also found. Therefore, upregulation of AKT3 in TKIs-treated resistant cells might be regulated by ubiquitination-proteasomal pathways. Elucidating the underlying mechanism is underway. Specifically targeting AKT3 by computer-aided drug discovery could tackle these existing clinical problems of lung adenocarcinoma harbouring EGFR activation mutation.

Cancer stem cells (CSCs) represent a population of cells within the tumor able to drive tumorigenesis and known to be highly resistant to conventional chemotherapy and radiotherapy. In this work, we show a new role for ETV7, a transcriptional repressor member of the ETS family, in promoting breast cancer stem-like cells plasticity and resistance to chemo- and radiotherapy in breast cancer (BC) cells. We observed that MCF7 and T47D BC-derived cells stably over-expressing ETV7 showed reduced sensitivity to the chemotherapeutic drug 5-Flouororuacil and to radiotherapy, accompanied by an adaptive proliferative behavior observed in different culture conditions. We further noticed that alteration of ETV7 expression could significantly affect the population of breast CSCs, measured by CD44⁺/CD24^low cell population and mammosphere formation efficiency. By transcriptome profiling, we identified a signature of Interferon-responsive genes significantly repressed in cells over-expressing ETV7, which could be responsible for the increase in the breast CSCs population, as this could be partially reverted by the treatment with IFN-beta. Lastly, we show that the expression of the IFN-responsive genes repressed by ETV7 could have prognostic value in breast cancer, as low expression of these genes was associated with a worse prognosis. We, therefore, suggest that an in-depth investigation of this mechanism could lead to novel breast CSCs targeted therapies and to the improvement of combinatorial regimens, possibly involving the therapeutic use of IFN-beta, with the aim of avoiding resistance development and relapse in breast cancer.

Colorectal cancer, a major cause of mortality worldwide, frequently metastasizes to the liver. Surgical resection of colorectal liver metastases (CLM), combined with systemic chemotherapy has the potential to be curative. However, CLM patients show heterogeneous clinical outcomes and degrees of response to therapy. Macrophages hold promise in the identification of clinically relevant markers to complement the current staging system in cancer and achieve a more refined patient stratification. In this work, we aimed to dissect the transcriptional heterogeneity of macrophages in human CLM and identify population-specific markers with clinical relevance. We performed single-cell RNA sequencing (scRNA-seq) of mononuclear phagocytes from CLM and normal adjacent liver, identifying a wide variety of tissue resident and tumor-enriched subsets of monocytes, dendritic cells and macrophages. A clear picture of the granularity within mononuclear phagocytes will allow us to evaluate the role of specific subpopulations in refractoriness to therapy. 

The selective dual inhibitor CDK4/6 (CDKi) in combination with endocrine therapy (ET) have increased progression-free survival (PFS) in luminal metastatic breast cancer (mBC) patients. Nevertheless, not all patients respond to polytherapy and those who respond, eventually develop resistance. We aim to decipher resistance and response mechanisms to CDKi plus ET therapy throughout the analysis of Circulating tumour cells (CTCs). CTCs were negative-enriched (RosetteSep, StemCell technologies) from 7.5 mL of blood at basal and after the first-cycle therapy. Pan-Cancer Panel (nCounter Technology) was performed in 6 luminal mBC patients, classified as responders and non-responders if resistance appeared 6 months after therapy initiation. Next, the selected genes were validated in 18 mBC patient´s samples by qRT-PCR. We observed that CTC expression analysis is a non-invasive alternative to decipher resistance mechanisms and therapy response in Luminal mBC patients treated with endocrine and CDKi therapy.  

Multi-kinase inhibitor resistance in glioblastoma cells can be reversed by reactivation of serine/threonine phosphatase 2A (PP2A) but the kinase targets for this synthetic lethal effect are ambiguous. To identify the target kinases, we established a Strategy for Characterization of Actionable Target kinase of multi-kinase inhibitors. Using this strategy, we revealed PI3K/AKT pathway and mitochondrial PDK kinases as targets for multi-kinase inhibitor UCN-01 in inducing synthetic lethality with pharmacological PP2A reactivation. Further, triplet combination of AKT inhibitor, PDK inhibitor, and PP2A reactivator was required for induction of synthetic lethality across glioblastoma cell models and showed significant therapeutic in vivo efficacy. In addition to glioblastoma, the findings were validated on medulloblastoma cells in vitro and in vivo. Collectively, we present a generalizable approach to identify specific actionable targets of multi-kinase inhibitors and demonstrate that triplet targeting of AKT, mitochondria and PP2A is required for inducing cytotoxicity across heterogeneous brain tumor cells.

   The epithelial-to-mesenchymal transition (EMT), which conveys epithelial (E) carcinoma cells to quasi-mesenchymal (qM) states, enables these cells to metastasize and acquire resistance to several drug and chemotherapeutic regimens. In addition, we have recently demonstrated that the EMT program also contributes to the generation of an immunosuppressive tumor microenvironment (TME) in breast carcinomas and confers resistance to immune checkpoint blockade therapies. Specifically, qM tumors recruit immunosuppressive cells to their TME and are refractory to checkpoint blockade, while E tumors recruit CD8+ T-cells instead and are sensitive to checkpoint blockade. Importantly, minority populations of qM cells can cross-protect their more E neighbors from immune attack. The underlying mechanisms by which this immunosuppression and cross-protection are achieved have been unclear. Using a combination of novel E and qM tumor models arising in syngeneic, immunocompetent hosts combined with CRISPR/Cas9 approaches, we demonstrate that qM but not E tumors are specifically associated with an immunosuppressive gene signature. Furthermore, abrogation of qM carcinoma cell-intrinsic factors (CD73, CSF1 or SPP1)  prevents the assembly of an immunosuppressive TME and sensitizes otherwise refractory qM tumors partially or completely to checkpoint blockade immunotherapy. Taken together, our work indicates that carcinoma cell-intrinsic factors specifically associated with residence in the qM state can directly influence their response to anti-tumor immunity and checkpoint blockade. As a result, this work brings to the forefront the possibility of using the epithelial-mesenchymal state of carcinoma cells as an important surrogate marker that can be used to predict responses to immune checkpoint blockade therapies.

Cancer relapse is due to a subset of malignant cells that resist treatment. We took advantage of an acute myeloid leukemia model: the PLZF-RARA retinoic acid (RA) resistant acute promyelocytic leukemia (APL) to characterize relapse-initiating cells and their vulnerabilities. By using an integrative single-cell multi-omics analysis (scRNA-seq and scATAC-seq), we uncovered transcriptional and chromatin heterogeneity of the PLZF-RARA APL blasts. We highlighted a subset of cells insensitive to RA-induced differentiation with a strong DNA repair and proliferation signature ("ReP" cluster) and exhibiting a fine-tuned transcriptional network targeting the histone methyltransferase Ezh2. Combining epigenomic profiling with mouse-derived models for Ezh2 catalytic inhibition or total KO, we revealed an independent methyltransferase Ezh2 activity linked to RA resistance. Thus, we unveil a dual role for Ezh2 in APL and suggest non-canonical targeting of Ezh2 to eliminate treatment-resistant leukemic cells.

Prostate cancer (PCa) is a leading cause of cancer-related mortality in men worldwide. Though PCa is known to be substantially heterogeneous at the molecular and histological level, there remains limited understanding of the tumor subpopulations (“clones”) generating intratumor heterogeneity (ITH) and their underlying dynamics. The impact of ITH on disease outcome remains unknown and a significant hurdle in treating PCa. This is especially evident in progression to castration resistant prostate cancer (CRPC): after initial androgen dependence, castration resistant clones emerge to populate the tumor, contributing to disease progression. Second generation androgen deprivation therapies have shown some success in treating CRPC, but eventually drug resistant clones emerge. Understanding the fundamental differences between clones may hold the key to improving therapies, preventing disease progression, and prolonging patient survival. Taking a multi-omics approach, we have mapped the clonal evolution during the transition to CRPC by whole organ mapping, single-cell sequencing, and RNAseq, with emphasis on transcriptomic changes. We have identified pathways and gene regulatory networks that control specific subpopulations as they become more castration resistant. Using pharmacogenomics and 3D organoid cultures, we have also identified potential novel drug resistance mechanisms and novel drug candidates. Our studies provide new mechanistic insights for identifying pre-existing high-risk clones with drug resistant phenotypes, uncovering novel therapeutic targets in CRPC. Our work has implications for designing new therapies aimed at controlling the emergence of advanced disease, preventing CRPC and drug resistance, with the ultimate goal of reducing mortality and improving prognosis.

Triple negative breast cancer (TNBC) is an aggressive sub-type of breast cancer which often lacks known targetable biomarkers. However, more than 75% of TNBC patients is associated with EGFR overexpression/amplification with a variable number of them showing a clinical benefit to EGFR-targeting therapies (1.7%-38.7%). Thus, suggesting EGFR dependency may represent a valuable vulnerability for a good portion of TNBCs. Here, we used a lentiviral barcode library to integrate a unique molecular mRNA barcode into each cell of an EGFR amplified TNBC cell-line to enable lineage-tracing. Next, by using scRNA-seq, we profiled more than 4,000 Afatinib tolerant cancer cells obtained by exposing them to an incremental concentration of Afatinib over time. Finally, we coupled machine learning with single-cell transcriptomics and lineage-tracing to identify genes whose expression is predictive of Afatinib response and develop a novel computational method to automatically predict response to Afatinib starting from the scRNA-seq of a TNBC.

Tumor heterogeneity is a key feature of melanomas that hinders the development of effective therapies. Aiming at finding common vulnerabilities, we identified LENOX (LincRNA-ENhancer of OXidative phosphorylation), a melanoma-specific lncRNA expressed in all known drug-tolerant states. LENOX expression was associated with poor prognosis in patients and its knock down induced melanoma cell apoptosis. By RAP-MS we identified RAP2C GTPase as a LENOX partner and found they co-localized at mitochondria. LENOX bridged a RAP2C-DRP1 interaction that inhibited DRP1 activity. LENOX or RAP2C knock-down thus increased mitochondrial fragmentation and impaired Oxphos. Upon MAP kinase pathway inhibition, LENOX promoted the metabolic switch from glycolysis to Oxphos, known to confer resistance to treatment. In line with this, LENOX or RAP2C silencing sensitized melanoma cells to apoptosis upon MAP kinase inhibition. We thus characterized a novel lincRNA-protein complex essential for mitochondrial function during melanoma progression, whose targeting may confer a long-lasting therapeutic response.

Patients with high-risk neuroblastoma (HR-NB) are treated with multimodal therapy, however approximately 60% will eventually suffer disease relapse and/or develop drug resistance. To date, the molecular mechanisms underlying drug resistance in NB are still greatly unknown. Our objective is to identify resistance-related signaling pathways in acquired chemoresistant HR-NB that can be targeted to improve therapeutic response. We generated in vitro drug-resistant NB cells to different cytotoxic drugs. By comparing gene expression profiles, we identified an acquired resistance-related signature. This gene signature showed enrichment for genes involved in cell differentiation, cell migration and DNA damage repair processes. Drug-resistant cells showed increased cell survival, improved invasion capacity and exhibited a significant increase of tumor growth when were injected in mice. Our findings contribute to a better understanding of the mechanism mediating treatment failure in NB. Further studies will be relevant to improve therapeutic response of drug-resistant NB.

We performed the present study to assess the Chinese herbal managing CID (chemotherapy induced drug resistance) in human pancreatic ductal adenocarcinoma. A retrospective observational clinical case was reported. Chemotherapeutic drug resistance is often accompanied with hospitalized cancer patients associated with anti-tumor therapy, so we called CID. In addition to the risk of tumor progression, the RWS (real world study) also showed the implementation of the originally scheduled chemotherapy with limits of dose and frequency. However, paying equal attention to Chinese and western medicine, CID therapy administered with Chinese herbal decoction may benefit those affected patients. We observed the curative effect of Chinese herbal Decoction of Warming and Removing(WRD) for CID (gemcitabine and cisplatin chemical drug resistance) in pancreatic ductal adenocarcinoma. Use of traditional CTM and western medicine symptomatic treatment, drug resistance partially overcome. Combination of Chinese and Western medicine ratio pure western medicine treatment the effect salience. Although few studies have been reported to demonstrate herbal ability to treat CID, our RWS suggested that Chinese herbal medicine might be helpful to overcome chemotherapy resistance in human pancreatic cancer. Chinese medicine has a good prospect in the treatment of CID, and it is of great value to be applied, and Chinese medicine also has the effect of anti-tumor, and has the advantages of multiple approaches, multiple targets, more efficacy and less side effects. Chinese herbal managing CID should be an additional choice with better benefits and tolerability in the treatment of CID in human pancreatic cancer.

Cancer represents one of the major health challenges worldwide. Solid tumours grow in a three-dimensional (3D) conformation that expose cells to very specific conditions, such as hypoxia and a heterogeneous distribution of nutrient levels, all characteristics that affect cell fate. Pancreatic cancer is an extremely lethal malignancy with a survival rate lower than any other cancer type. Bioengineering systems, such as three-dimensional patient-specific models, are progressively emerging as systems better able to mimic the biology of pancreatic tumors and to test new anticancer therapies, as they more efficiently recapitulate the complex tumor microenvironment characteristic of pancreatic tumors. In our lab, we developed a hanging drop plus methylcellulose technique to optimize the long-term culture and manipulation of human pancreatic tumours cells, characterized by low cohesiveness and low manageability, using MiaPaCa-2 cell line. Moreover, the acidification of the tumour microenvironment is a prominent environmental driver of cancer progression. Within this context, we developed efficient optical ratiometric microparticles suitable to detect intra- or extracellular pH changes in in vitro cell cultures. By means of time-lapse confocal microscopy we were able to generate spatially resolved maps of pH across the 3D cultures of human mesenchymal stem cells. More recently, we developed a novel methodology to successfully couple the ratiometric dye pyranine with silica microparticles. The resulting pH-sensing microparticles show superior pH sensitivity and good biocompatibility, two key properties for their applications as pH-sensors in living systems.

Dysregulation of kinase signaling pathways via mutations favors tumor cell survival and resistance to therapy. Here, we reveal a novel mechanism of kinase signaling regulation and nuclear receptor activity via deubiquitination. We observed that ubiquitin-specific protease 7 (USP7) and 11 (USP11) are highly expressed in acute leukemia patients with high-risk disease, forming a complex to deubiquitinate the oncogenic lymphocyte cell-specific protein-tyrosine kinase (LCK). Deubiquitination of LCK controls its activity, altering T cell receptor signaling. Impairment of LCK activity leads to increased expression of the glucocorticoid receptor transcript, increased transcription of pro-apoptotic target genes, such as BCL2L11, and sensitizes cells to glucocorticoids in primary T cell leukemia patient samples. This transcriptional activation is orchestrated by the deubiquitinase activity and mediated via an increase in enhancer-promoter interaction intensity. Our data unveil how dysregulated deubiquitination controls signaling pathways leading to cancer cell survival, drug non-response, and suggest novel therapeutic combinations targeting leukemia.

Many cancer subtypes harbor mutations of the mitogen-activated protein kinase (MAPK) pathway, offering continuous access to cell-cycle entry and unfettered tumor proliferation. Modern small molecule therapies for such cancers target critical kinase members of the MAPK signaling cascade (EGFR, RAS, RAF, MEK), all of which are highly effective at preventing cell-cycle activity. These targeted therapies at high doses and in combinations, however, fail to completely halt proliferative activity within the first week in drug, as a subpopulation of cancer cells can escape drug action. We found this behavior to be true in a variety of cancer cell types harboring BRAF^V600E, KRAS^G12C, or EGFR^E746-A750del mutations. Further, the cell cycles performed by these drug-escaping subpopulations were all significantly lengthened with indications of DNA under-replication, yet often ended in successful mitoses and DNA damage accumulation, as seen by high-throughput time-lapse microscopy. We found that these escapee cells were able to tolerate such stressful cell cycles by initiating (i) DNA replication recovery mechanisms facilitated by FANCD2 recruitment, and (ii) a widely cytoprotective stress response via transcription factor ATF4. The culmination of such protective pathways results in an escapee cell’s heightened probability to avoid apoptosis and complete its mitotic division with presumed greater genomic instability. Together, these findings highlight the need for more complete and durable responses in the clinic for MAPK pathway inhibitors, which will likely be achieved through the suppression of unique stress tolerability in treated tumors.

We demonstrate positive staining of a protein kinase CK2α in nucleoli was strongly associated with recurrence and poor patient outcomes. In this study, we employed IHC staining by specimens from invasive ductal carcinomas (IDC) of the breast to evaluate CK2α protein levels and subcellular localization. The association of nucleolar CK2α with poor prognosis among the triple-negative (p=0.0069) and late-stage (p Stage 3, p=0.0073) breast cancers was evident, in which RFS decreased to roughly 50% in nucleolar CK2α-positive patients. We propose that IHC evaluation of nucleolar CK2α-positive staining may be a new and independent prognostic factor (HR=5.264, p=0.017 in the multivariate analysis). The application of this analysis will contribute to the early decision of treatment strategy and to companion diagnostics, following surgical resection of the tumor. A more detailed evaluation of CK2α function in nucleolar events in malignant cells may lead to the development of novel anti-cancer therapeutics.

IDH1 mutations are implicated in Acute Myeloid Leukemia (AML) through abnormal production of oncometabolite 2-hydroxyglutarate causing widespread changes in DNA methylation. While primary or acquired resistance to IDH inhibitor therapies represents a major problem, mechanisms are poorly understood. We combine gene expression, Transcription Factor (TF) activity, and clinical outcome in patients to uncover pathways related to IDH inhibitor resistance. We studied gene expression in 64 patients with relapsed or refractory AML receiving IDH inhibitor therapy at diagnosis and relapse. TF activities showed that RUNX1 and CEBPa activities are linked to the clinical outcome. Other TFs significantly differentially inactive in complete remission samples confirm that leukemia stemness is important in primary resistance. Analyses using chromatin structure data are in progress to link the regulation of these TFs to epigenetic mechanisms impacted by IDH mutation. Mitochondrial metabolism is an important resistance pathway in AML and these results confirm its connection to stemness. 

Radiotherapy is a potent modality for breast cancer treatment. However, surviving radioresistant cells can cause tumor relapse after radiotherapy. We exposed MCF7 breast cancer cells to daily doses of 2 Gy radiation up to an accumulated dose of 20 Gy, and compared the surviving cells to age-matched sham-irradiated cells and the parental MCF7 cells. Fractionally irradiated cells (FIR20) displayed increased clonogenic survival and population doubling time. RNA sequencing revealed significant changes in the levels of 229 mRNAs and 7 circRNAs between FIR20 and both controls. Downstream pathway analysis demonstrated among others repressed estrogen response signaling and cell cycle regulation in FIR20. We found evidence of cross-resistance to endocrine therapies, cancer stem cell-like plasticity, and luminal to basal-like subtype plasticity. Lastly, we elucidated several candidate regulatory circRNA-miRNA-mRNA interactions. Our study reveals potential mechanisms of radioresistance and therapeutic modules for targeting radioresistant breast cancer cells.

Resistance to targeted therapies is a significant clinical problem in HER2-positive (HER2+) breast cancer. “Drug-tolerant persisters” (DTPs), a sub-population of cancer cells that survive via reversible, non-genetic mechanisms, are implicated in resistance to tyrosine kinase inhibitors (TKIs) in other malignancies, but DTPs following HER2 TKI exposure have not been well characterized. We found that HER2 TKIs (lapatinib, tucatinib) evoke DTPs with either a luminal-like or a mesenchymal-like transcriptome. Lentiviral barcoding/single cell RNA-sequencing reveal that HER2+ cells cycle stochastically through a “pre-DTP” state, characterized by a G₀-like expression signature and enriched for diapause and/or senescence genes. Trajectory analysis/cell sorting show that pre-DTPs preferentially yield DTPs upon HER2 TKI exposure. Cells with similar transcriptomes are seen in HER2+ tumors and are associated with poor TKI response. Finally, biochemical experiments indicate that luminal-like DTPs survive via estrogen receptor-dependent induction of SGK3, leading to rewiring of the PI3K/AKT/mTORC1 pathway to enable AKT-independent mTORC1 activation.

Hepatocellular carcinoma (HCC) is one of the deadliest cancers with limited therapeutic opportunities. Sorafenib, the predominant clinical drug for advanced HCC provides limited extension in median overall survival due to resistance. We show that nutrient withdrawal can reinstate sorafenib sensitivity in resistant HCC cells, xenografts and patient-derived HCC organoids. Sorafenib inhibits mitochondrial respiration, causing resistant HCC cells to switch to glycolysis for survival. Synergistically, reduced nutrients prevent this Warburg shift leading to sensitisation to sorafenib in vitro and in vivo. Detailed functional experiments show that glucose is the limiting nutrient crucial for curtailing this metabolic flexibility. We further show that p53 is necessary and sufficient for the sorafenib-sensitizing effect of fasting, as p53 knock out cells remain resistant under starvation. p53 is also necessary for the sorafenib-enhancing effect of fasting in an orthotopic HCC mouse model. Our data indicate fasting and sorafenib as a potential polytherapy for resistant advanced-stage HCC.

Immunogenic cell death (ICD) in malignant cells can decrease tumor burden and activate anti-tumor immune response to obtain lasting anti-tumor immunity, leading to the elimination of distant metastases and prevention of recurrence. Here, we reveal that ppM1 peptide is capable of forming irreparable transmembrane pores on tumor cell membrane, leading to ICD which we name poroptosis. Poroptosis is directly dependent on cell membrane nanopores regardless of the upstream signaling of cell death. ppM1-induced poroptosis was characterized by the sustained release of intracellular LDH. This unique feature is distinct from other well-characterized types of acute necrosis induced by freezing- thawing (F/T) and detergents, which leads to the burst release of intracellular LDH. Our results suggested that steady transmembrane-nanopore-mediated subacute cell death played a vital role in subsequent activated immunity that transforms to an antitumor immune microenvironment. Selectively generating poroptosis in cancer cell could be a promise strategy for cancer therapy. 

Activating splice site mutations in the MET proto-oncogene (METΔex14) have emerged as a therapeutic target in lung cancer. However, past experience with targeted therapies against other drivers (e.g. EGFR) point to the high likelihood of tumours acquiring drug resistance. To identify targets for overcoming therapeutic resistance, we created drug-resistant clones from METΔex14-expressing cancer cells. Targeted sequencing identified mutations in SPOP and MGA in two independent clones, with both targets being negative regulators of MYC stability and activity. This finding was supported by the observation that MYC levels were higher in resistant lines harbouring these mutations. In parallel, transcriptome analysis of our resistant clones revealed a distinct MYC activation signature. Finally, we found BRD4, a targetable epigenetic regulator of MYC activity, to be upregulated in resistant clones. These findings converge on MYC as a likely mediator of resistance in MET TKI-resistant cancers, offering a potential therapeutic window for countering MYC-mediated resistance.

Despite clinical efficiency of therapies targeting cell-surface receptor ErbB-2, resistance to such drugs is a major issue in breast cancer (BC). ErbB-2 triggers oncogenic signaling when is membrane-bound, but it also migrates to the nucleus (NErbB-2) where it acts as a transcription regulator. Here, we explored ErbB-2 induced transcriptome using a resistant model of BC with high NErbB-2 levels. Eviction of NErbB-2 modulated the expression of genes involved in type-I interferon signaling pathway. Preclinical assays demonstrated that blockade of NErbB-2 abrogates tumor growth and induced expression of intermediary genes such as IFNB1, OAS2, and TRIM22. Analysis of local chromatin architecture identified ErbB-2 constitutive recruitment onto IFNB1 regulatory regions, while its nuclear eviction resulted in high levels of histone H4 acetylation, a marker of chromatin activation. These results revealed repression of type-I interferon pathway as a mechanism of carcinogenesis in ErbB-2-positive BC highlighting NErbB-2 as a therapeutic target in resistant BC.

Chemotherapy is a common treatment for prostate cancer (PCa) but in which sometimes patients present the development of resistance. NRF2 is a key protein involved with drug efflux, detoxification, and oxidative stress balance. Cancer cells submitted to chemotherapy often promote NRF2 activation to benefit themselves with the cytoprotective response mediated by this protein. Here, we performed the NRF2 knockout(NKO) in DU-145 and PC-3 PCa cells, through CRISPR/Cas9, which knockout was confirmed through Sanger sequencing and downregulation of targets,NQO1 and HO1. PC3-NKO cells exhibited higher levels of ROS compared to wild-type. Despite no modulation in ROS content was observed between DU145-NKO and wild-type cells, DU145-NKO cells seem to be more sensitive to the treatment with docetaxel, while to PC3-NKO cells weren’t observed differences in IC50 values. This data showed that NRF2 can be a potential target to sensitize cancer cells to chemotherapy but depending on the PCa cell profile.

Medulloblastoma (MB) is the most common malignant brain tumor of childhood. Despite high-dose radio/chemotherapy treatment, 15-30% of patients still display a high risk of tumor recurrence. 

In order to understand the mechanism that sustain MB chemotherapy resistance, we setup an in vitro model of MB drug resistance by exposing patient-derived MB cells to a combination of commonly used chemotherapeutics for pediatric MB treatment.

The transcriptomic and proteomic characterization of parental and drug-resistant MB cells revealed i) the activation of neurotrophin and inflammatory signaling, ii) the downregulation of neuronal differentiation markers and, iii) the alterations of protein folding, mitochondrial activity and cell metabolism. Finally, High-Throughput drug Screening (HTS) identified the antimetabolites as class of drugs particularly active against refractory MB models with a remarkable synergistic effect with standard chemotherapy.

The integration of omics data together with their functional validation through interrogation of HTS results, highlighted the metabolic reprogramming as a distinctive hallmark of MB resistant cells.

The main aim of our study is to determine mechanisms of resistance to sunitinib and sorafenib in ccRCC. Our results indicate two, very different mechanisms, but both lead to similar effects. Cells treated with sorafenib undergo epithelial to mesenchymal transition, with enhanced migration rate, upregulation of MMP9 and changes in morphology. While sunitinib treatment results in cell senescence, upregulation of e-cadherin and secretion of pro-angiogenic factors. Moreover, sunitinib and sorafenib resistant tumors were characterized by better developed vascularity with high levels of blood vessel markers, compared to control. Stimulation of endothelial cells with conditioned media from resistant Caki-1 cells resulted in disruption of ECs monolayer integrity due to phosphorylation and internalization of VE-cadherin.

Our results suggest that despite of multi-target inhibition, tumor cells are able to compensate inactivation of pathways involved in angiogenesis which leads in consequence to increased aggressiveness and survival of those cells and activation of ECs.

Residual disease, remains one of the major clinical challenges towards full cure. In triple-negative breast cancers, persistence to chemotherapy results in the highest risk of recurrence among all breast cancer subtypes. If there is now consensus that the drug-tolerant state is defined by non-genetic features, understanding the mechanisms driving tumor cells to drug-tolerance stands as a pre-requisite to efficiently fight recurrence. Here, we showed that the repressive histone mark H3K27me3 is a determinant of cell fate at the onset of chemotherapy exposure. We demonstrated that (i) the persister expression program is primed with both H3K4me3 and H3K27me3 in unchallenged cells, H3K27me3 being the lock to its transcriptional activation, (ii) H3K27me3 controls cell fate upon chemotherapy exposure: alleviating the genome from H3K27me3 enhances the potential of each cancer cell to tolerate chemotherapy insult. Our results highlight how chromatin landscapes shape the potential of cancer cells to respond to initial therapeutic insult.

High grade serous ovarian carcinoma (HGSOC) is the most diagnosed and lethal type of ovarian cancer. Half of HGSOC present mutations in BRCA 1, BRCA 2 or other genes related to the homologous recombination repair pathway (HR). In order to maintain their homeostasis, HR deficient tumours present a higher dependence on other repairing mechanisms, such as those dependent of PARP activity. To maintain PARP activated, the tumour has to change its metabolism to a more oxidative one, to provide high amounts of NAD⁺, which is PARP’s substrate. Our results indicate that tumours displaying a more glycolytic metabolism present more resistance to PARP inhibitors (PARPi). In consequence, the combination of OXPHOS inhibitors (such metformin), which increased glycolytic metabolism, with PARP inhibitors in HR-defective cells exhibit a reduced effect. In addition, we have observed that HGSOC patients with BRCA mutations that are resistant to PARPi present higher expression of glycolytic markers and lower expression of OXPHOS markers. Hence, these markers could be used to predict the tumour response to PARPi. Using drugs that increase OXPHOS metabolism could be used as an enhancer of PARPi effects, especially in resistant tumours.

High-Grade Serous Ovarian Cancer, the most aggressive subtype of epithelial ovarian cancer, has an overwhelming presence of TP53 mutations in 96% cases along with amplifications in PI3K/AKT pathway genes. Recently, we highlighted the role of serine-46 phosphorylation, a post-translational modification of Wtp53, in attenuating PIK3CA activity. Although, TCGA data revealed association of p53 mutations with upregulated PIK3CA expression, the underlying mechanisms of regulation have not been investigated. Thus, we expressed p53 hotspot mutants in p53-null cells wherein they exhibited differential cisplatin and alpelisib sensitivity, altered phosphorylation patterns, differential ERK/AKT activation and p53 target gene expression. Interestingly, mutant-R282W, with highest resistance to cisplatin and alpelisib and increased cisplatin-induced PIK3CA expression, showed sensitivity to combinatorial treatment. Additionally, alpelisib pre-treatment in relapsed patient-derived cells, sensitized them to subsequent cisplatin treatment. Thus, investigating association of mutant-p53 and its PTMs with PIK3CA pathway will pave the way for alpelisib-platinum therapy in relapsed ovarian cancer.

Metabolomics can uncover complex metabolic signatures of drug treatment responses and could be extremely valuable to investigate treatment efficacy. Our group previously developed a long-term ovarian cancer (OvC) patient-derived ex vivo model, in which original tumor features and tumor microenvironment components are retained. Here, our goal is to combine data from 9 OvC ex vivo samples, namely the cellular drug response and changes in the metabolic footprint to build a predictive model of drug efficacy. We challenged the cultures with the standard-of-care chemotherapeutics carboplatin and paclitaxel, alone or in combination. We observed an ex vivo sample-specific drug response and the exploratory analysis of metabolic footprints revealed different groups among ex vivo samples and treatments, supporting the existence of sample and treatment-associated metabolic signatures. Machine learning approaches were used to explore the relationship between ex vivo drug response and metabolic footprints as a step towards patient stratification in the clinical setting.

Tumor response to checkpoint immunotherapy (IT) can be enhanced by radiation, whereas immunosuppressive factors acquired by radioresistant (RR) cancer cells may severely compromise the IT efficacy. Here, we reveal that RR glioblastoma (GBM) cells enriched with GBM stem cells can boost lipid metabolism not only to fuel aggressive growth but also to escape immune attack by CD47 expression.  Poor clinic outcomes among recurrent GBM patients are linked to the co-expression of three fatty acid oxidation (FAO) enzymes (CPT1A, CPT2 and ACAD9) and CD47 which is detected in the tumors re-grown after radiation. By inhibition of FAO or CRISPR-knockout of FAO genes, we demonstrate that FAO-derived citrates promote CD47 transcription via acetylation of NF-κB/RelA. Blocking FAO enhances macrophage-phagocytosis and impairs neurosphere formation; dual targeting of FAO and CD47 diminishes orthotopic tumor regrowth with intensified antibody penetration and macrophage-phagocytosis. Thus, targeting FAO-CD47 axis may improve GBM control by eliminating the radioresistant-anti-phagocytotic cancer cells.      

Non-small cell lung cancer (NSCLC) comprises 80% to 85% of lung cancer, and due to the intrinsic resistance to chemotherapy, the survival rates of NSCLC patients remain unimproved. Deciphering the pathogenesis of signaling pathways contributing to drug resistance can help identify efficient drug combinations for enhancing targeted therapies. In our study, we describe an image-based multiplexing approach, rapid multiplexed immunofluorescence (RapMIF) to decipher the cell-to-cell heterogeneity in signaling networks. RapMIF automated iterative staining, bleaching, and imaging processes, and revealed the spatial networks of two signaling pathways that may contribute to the development of drug resistance, WNT/β-catenin, and AKT/mTOR pathways. The multiplex data showed protein translocation, subcellular signaling clusters, and crosstalk between pathways at a single-cell resolution. The spatial signaling was also demonstrated in drug-responsive cultures, EGFR-mutant drug-sensitive cells in response to osimertinib, EGFR TKI. RapMIF provides a framework for identifying reliable biomarkers and for precision drug design.