Temozolomide-Acquired Resistance Is Associated with Modulation of the Integrin Repertoire in Glioblastoma, Impact of α5β1 Integrin

Despite extensive treatment, glioblastoma inevitably recurs, leading to an overall survival of around 16 months. Understanding why and how tumours resist to radio/chemotherapies is crucial to overcome this unmet oncological challenge. Primary and acquired resistance to Temozolomide (TMZ), the standard-of-care chemotherapeutic drug, have been the subjects of several studies. This work aimed to evaluate molecular and phenotypic changes occurring during and after TMZ treatment in a glioblastoma cell model, the U87MG. These initially TMZ-sensitive cells acquire long-lasting resistance even after removal of the drug. Transcriptomic analysis revealed that profound changes occurred between parental and resistant cells, particularly at the level of the integrin repertoire. Focusing on α5β1 integrin, which we proposed earlier as a glioblastoma therapeutic target, we demonstrated that its expression was decreased in the presence of TMZ but restored after removal of the drug. In this glioblastoma model of recurrence, α5β1 integrin plays an important role in the proliferation and migration of tumoral cells. We also demonstrated that reactivating p53 by MDM2 inhibitors concomitantly with the inhibition of this integrin in recurrent cells may overcome the TMZ resistance. Our results may explain some integrin-based targeted therapy failure as integrin expressions are highly switchable during the time of treatment. We also propose an alternative way to alter the viability of recurrent glioblastoma cells expressing a high level of α5β1 integrin.

Loss of GdpP function in Staphylococcus aureus leads to β-lactam tolerance and enhanced evolution of β-lactam resistance

Infections caused by Staphylococcus aureus are a leading cause of mortality. Treating infections caused by S. aureus is difficult due to resistance against most traditional antibiotics, including β-lactams. We previously reported the presence of mutations in gdpP among S. aureus strains that were obtained by serial passaging in β-lactam drugs. Similar mutations have recently been reported in natural S. aureus isolates that are either non-susceptible or resistant to β-lactam antibiotics. gdpP codes for a phosphodiesterase that cleaves cyclic-di-AMP (CDA), a newly discovered second messenger. In this study, we sought to identify the role of gdpP in β-lactam resistance in S. aureus . Our results showed that gdpP associated mutations caused loss of phosphodiesterase function, leading to increased CDA accumulation in the bacterial cytosol. Deletion of gdpP led to an enhanced ability of the bacteria to withstand a β-lactam challenge (two to three log increase in bacterial colony forming units) by promoting tolerance without enhancing MICs of β-lactam antibiotics. Our results demonstrated that increased drug tolerance due to loss of GdpP function can provide a selective advantage in acquisition of high-level β-lactam resistance. Loss of GdpP function thus increases tolerance to β-lactams that can lead to its therapy failure and can permit β-lactam resistance to occur more readily.

Spatial protein heterogeneity analysis in frozen tissues to evaluate tumor heterogeneity

A new workflow for protein-based tumor heterogeneity probing in tissues is here presented. Tumor heterogeneity is believed to be key for therapy failure and differences in prognosis in cancer patients. Comprehending tumor heterogeneity, especially at the protein level, is critical for tracking tumor evolution, and showing the presence of different phenotypical variants and their location with respect to tissue architecture. Although a variety of techniques is available for quantifying protein expression, the heterogeneity observed in the tissue is rarely addressed. The proposed method is validated in breast cancer fresh-frozen tissues derived from five patients. Protein expression is quantified on the tissue regions of interest (ROI) with a resolution of up to 100 μm in diameter. High heterogeneity values across the analyzed patients in proteins such as cytokeratin 7, β-actin and epidermal growth factor receptor (EGFR) using a Shannon entropy analysis are observed. Additionally, ROIs are clustered according to their expression levels, showing their location in the tissue section, and highlighting that similar phenotypical variants are not always located in neighboring regions. Interestingly, a patient with a phenotype related to increased aggressiveness of the tumor presents a unique protein expression pattern. In summary, a workflow for the localized extraction and protein analysis of regions of interest from frozen tissues, enabling the evaluation of tumor heterogeneity at the protein level is presented.

Propensity Score Matching Analysis Comparing Ruxolitinib Vs Historical Controls in 2 nd Line or Beyond Treatment for Chronic Gvhd after Therapy Failure

Background The REACH3 study has reported greater overall response and failure-free survival (FFS) with Ruxolitinib (RUX) as 2 nd line therapy for steroid-refractory chronic graft versus host disease (cGVHD) in comparison to best available therapy (BAT; NEJM 2021). In addition, our Canadian retrospective study as a real-world experience has reported a promising activity of 64.6% FFS and 83.3% overall survival (OS) at 12 months in 115 patients (pts) treated with RUX from 2016 to 2021 for heavily pretreated cGVHD (ASH 2021). Also, 47.6% pts could reduce corticosteroid dose below 0.1mg/kg/day within 6 months. The present study compared treatment outcomes using a propensity-score matching (PSM) analysis between RUX pts ("RUX group", n=115) and a historical cohort of cGVHD pts treated with BAT as 2 nd line therapy or beyond from 2005 to 2013 ("BAT group", n=311). Statistical endpoints such as FFS, OS and steroid dose reduction were evaluated instead of overall response due to limited response assessment available from retrospective chart reviews. Patients and methods BAT included mycophenolate (43%), prednisone (29%), prednisone/cyclosporine (12%), extracorporeal photopheresis (6%), rituximab (3%), and others (7%). The pts and disease characteristics between the RUX vs BAT groups were not well balanced, as expected: RUX group showed a higher number of pts with severe grade GVHD (59.1% in RUX vs. 20.3% in BAT; p<0.0001), and more heavily pretreated with 4 th line or beyond (84.3% in RUX vs. 17.0% in BAT; p<0.0001). FFS and OS were calculated from the day of starting RUX or BAT therapy, while daily prednisone doses at months 0, 3 and 6 were calculated divided by body weight (kg). PSM analysis was applied to adjust risk factors that were unbalanced between the 2 groups, including GVHD severity at therapy start (mild/moderate vs severe grade), HCT-CI score (0-2 vs 3 or beyond), and treatment line (2nd vs 3rd vs 4th line or beyond). A total of 100 patients (i.e. 50 case-control pairs) were finally extracted through PSM process within 0.2 of calliper difference. PSM analysis balanced out the risk factors between the 2 groups: GVHD severity (p=1.0), HCT-CI score (p=1.0) and treatment line (p=1.0). The FFS and OS rate at 12 months were compared using Cox's proportional hazard model. Results In the overall population (n=426), with a median 19 months follow-up duration, 244 failures (57.3%) were noted. While both groups showed similar failure rates due to non-relapse mortality (NRM), the RUX group showed significantly lower failure rate from cGvHD resistance requiring therapy switch. Failure was noted in 40 pts (33.4%) in RUX due to resistance requiring a switch of therapy (n=23; 20.0%), NRM (n=14, 12.2%) and relapse (n=3; 2.6%), while failure occurred in 204 pts (65.6%) in BAT due to resistance requiring a switch of therapy (n=142; 45.7%), NRM (n=36; 11.6%) and relapse (n=26; 12.7%). The 12 months' FFS rate were 64.7% and 40.1% in RUX vs. BAT group (p<0.0001; Fig 1A), while the 12 months' OS rate were 83.4% and 83.7% in RUX vs. BAT group (p=0.913; Fig 1B). In the propensity score matched (PSM) cohort (n=100), the RUX group showed a survival benefit over the BAT group: the 12 months' FFS rate was 74.0% and 29.7% in RUX vs. BAT group (p<0.0001; Fig 2A), while the 12 months' OS rate were 90.5% and 80.2% in RUX vs. BAT group (p=0.109; Fig 2B). Multivariate analysis in the PSM cohort confirmed that RUX is superior to BAT for FFS (p<0.001, HR 0.267 [0.139-0.516]) together with HCT-CI score 0-2 vs ≥3 (p=0.037, HR 0.490 [0.251-0.959]) with a trend to better survival in RUX over BAT (p=0.110, HR 0.402 [0.131-1.228]). The prednisone dose was gradually reduced over time: the median doses of prednisone at months 0, 3, and 6 were 0.34, 0.16 and 0.02 mg/kg/day in the RUX group, and 0.95, 0.24 and 0.15 mg/kg/day at months 0, 3 and 6 in the BAT group. RUX facilitates the discontinuation of prednisone. The proportions of patients who discontinued prednisone at months 0, 3 and 6 were 8.7%, 15.9% and 24.1% in RUX, and 0.7%, 0% and 2.0% in BAT group. The differences in proportions of pts that discontinued prednisone between the RUX vs BAT groups at months 0, 3, and 6 were 8.0%, 15.9% and 22.1% (Fig 3). Conclusion: The current PSM analysis study suggests that RUX showed a superior FFS to BAT as 2nd line therapy or beyond in cGVHD patients after therapy failure. RUX showed better steroid tapering compared to BAT. Figure 1 Figure 1. Disclosures White: Novartis: Honoraria. Elemary: Pfizer, Janssen: Membership on an entity's Board of Directors or advisory committees; Jazz, BMS, Abbvie, Novartis, Pfiz: Membership on an entity's Board of Directors or advisory committees. Hamad: Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Law: Novartis: Consultancy; Actinium Pharmaceuticals: Research Funding. Kim: Bristol-Meier Squibb: Research Funding; Paladin: Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Honoraria, Research Funding.

CSIG-04. UNCOUPLING OF ETS1 FROM MAPK PATHWAY SIGNALS AS RESISTANCE MECHANISM TOWARDS BRAF INHIBITORS IN BRAF-MUTATED CHILDHOOD GLIOMA

BACKGROUND High-grade gliomas are among the most aggressive brain tumors across all age groups. BRAF is within the most frequently altered genes in pediatric glioma, sometimes connected with telomerase reverse transcriptase (TERT) promoter mutations, predicting a particularly aggressive course of disease. Precision medicine approaches targeting the MAPK pathway have shown promising results in patients with BRAF-mutated glioma. Although acquired insensitivity to BRAF inhibitors appears as major issue for therapy failure, underlying molecular mechanisms are still poorly understood. METHODS Cell models from an anaplastic pleomorphic xanthoastrocytoma with BRAF V600E and TERT promoter mutations and the recurrent tumor, operated following MAPK pathway-targeting therapy, were established. Furthermore, a dabrafenib-resistant subline of the recurrent tumor was generated. The patient-derived cell models were genetically characterized using array-based genomic hybridization (aCGH). Sensitivity of the cells towards different MAPK pathway inhibitors was tested. Basal expression and activation of MAPK pathway and downstream signals were analyzed by qRT-PCR and Western blots. RESULTS Screening a panel of both primary and immortalized glioma cell models with different BRAF and TERT promoter status revealed significantly induced MAPK pathway activation and enhanced TERT levels in BRAF V600E and TERT promoter double-mutant gliomas. Furthermore, cells with both mutations were hyper-sensitive towards BRAF-targeting agents and BRAF inhibition resulted in reduced TERT levels. ETS1 expression was strongly increased in the recurrent tumor and identified as important player in telomerase re-activation. aCGH revealed gains of chromosomal regions encoding for different ETS-factors in the dabrafenib-resistant subline. Western blot analyses suggested a BRAF/ERK-independent survival mechanism in the dabrafenib-resistant subline. Accordingly, dabrafenib insensitivity triggered cross-resistance towards the MEK inhibitor trametinib. Uncoupled from the MAPK pathway, ETS1 expression was further upregulated in the dabrafenib-resistant subline. CONSLUSION: Taken together, our data demonstrate that MAPK-independent ETS transcription factor upregulation is a central mechanism of BRAF inhibitor therapy failure in BRAF-mutated pediatric glioma patients.

p32 promotes melanoma progression and metastasis by targeting EMT markers, Akt/PKB pathway, and tumor microenvironment

Melanoma originates from melanin-producing cells called melanocytes. Melanoma poses a great risk because of its rapid ability to spread and invade new organs. Cellular metastasis involves alteration in the gene expression profile and their transformation from epithelial to mesenchymal state. Despite of several advances, metastatic melanoma being a key cause of therapy failure and mortality remains poorly understood. p32 has been found to be involved in various physiological and pathophysiological conditions. However, the role of p32 in melanoma progression and metastasis remains underexplored. Here, we identify the role of p32 in the malignancy of both murine and human melanoma. p32 knockdown leads to reduced cell proliferation, migration, and invasion in murine and human melanoma cells. Furthermore, p32 promotes in vitro tumorigenesis, inducing oncogenes and EMT markers. Mechanistically, we show p32 regulates tumorigenic and metastatic properties through the Akt/PKB signaling pathway in both murine and human melanoma. Furthermore, p32 silencing attenuates melanoma tumor progression and lung metastasis in vivo, modulating the tumor microenvironment by inhibiting the angiogenesis, infiltration of macrophages, and leukocytes in mice. Taken together, our findings identify that p32 drives melanoma progression, metastasis, and regulates the tumor microenvironment. p32 can be a target of a novel therapeutic approach in the regulation of melanoma progression and metastasis.

In Vivo Secretion of β-Lactamase-Carrying Outer Membrane Vesicles as a Mechanism of β-Lactam Therapy Failure

Outer membrane vesicles carrying β-lactamase (βLOMVs) protect bacteria against β-lactam antibiotics under experimental conditions, but their protective role during a patient’s treatment leading to the therapy failure is unknown. We investigated the role of βLOMVs in amoxicillin therapy failure in a patient with group A Streptococcus pyogenes (GAS) pharyngotonsillitis. The patient’s throat culture was examined by standard microbiological procedures. Bacterial vesicles were analyzed for β-lactamase by immunoblot and the nitrocefin assay, and in vivo secretion of βLOMVs was detected by electron microscopy. These analyses demonstrated that the patient’s throat culture grew, besides amoxicillin-susceptible GAS, an amoxicillin-resistant nontypeable Haemophilus influenzae (NTHi), which secreted βLOMVs. Secretion and β-lactamase activity of NTHi βLOMVs were induced by amoxicillin concentrations reached in the tonsils during therapy. The presence of NTHi βLOMVs significantly increased the minimal inhibitory concentration of amoxicillin for GAS and thereby protected GAS against bactericidal concentrations of amoxicillin. NTHi βLOMVs were identified in the patient’s pharyngotonsillar swabs and saliva, demonstrating their secretion in vivo at the site of infection. We conclude that the pathogen protection via βLOMVs secreted by the flora colonizing the infection site represents a yet underestimated mechanism of β-lactam therapy failure that warrants attention in clinical studies.