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Pancreatic cancer is an increasing cause of cancer related death worldwide. KRAS is the dominant oncogene in this cancer type and molecular rationale would indicate, that inhibitors of the downstream target MEK could be appropriate targeted agents, but clinical trials have failed so far to achieve statistically significant benefit in unselected patients. We aimed to identify predictive molecular biomarkers that can help to define subgroups where MEK inhibitors might be beneficial alone or in combination. Next-generation sequencing data of 50 genes in three pancreatic cancer cell lines (MiaPaCa2, BxPC3 and Panc1) were analyzed and compared to the molecular profile of 138 clinical pancreatic cancer samples to identify the molecular subtypes of pancreatic cancer these cell lines represent. Luminescent cell viability assay was used to determine the sensitivity of cell lines to kinase inhibitors. Western blot was used to analyze the pathway activity of the examined cell lines. According to our cell viability and pathway activity data on these model cell lines only cells harboring the rare G12C KRAS mutation and low EGFR expression are sensitive to single MEK inhibitor (trametinib) treatment. The common G12D KRAS mutation leads to elevated baseline Akt activity, thus treatment with single MEK inhibitors fails. However, combination of MEK and Akt inhibitors are synergistic in this case. In case of wild-type KRAS and high EGFR expression MEK inhibitor induced Akt phosphorylation leads to trametinib resistance which necessitates for MEK and EGFR or Akt inhibitor combination treatment. In all we provide strong preclinical rational and possible molecular mechanism to revisit MEK inhibitor therapy in pancreatic cancer in both monotherapy and combination, based on molecular profile analysis of pancreatic cancer samples and cell lines. According to our most remarkable finding, a small subgroup of patients with G12C KRAS mutation may still benefit from MEK inhibitor monotherapy.

For a long time necrosis was thought to be an uncontrolled process but evidences recently have revealed that necrosis can also occur in a regulated manner. Necroptosis, a type of programmed necrosis is defined as a death receptor-initiated process under caspase-compromised conditions. The process requires the kinase activity of receptor-interacting protein kinase 1 and 3 (RIPK1 and RIPK3) and mixed lineage kinase domain-like protein (MLKL), as a substrate of RIPK3. The further downstream events remain elusive. We applied known inhibitors to characterize the contributing enzymes in necroptosis and their effect on cell viability and different cellular functions were detected mainly by flow cytometry. Here we report that staurosporine, the classical inducer of intrinsic apoptotic pathway can induce necroptosis under caspase-compromised conditions in U937 cell line. This process could be hampered at least partially by the RIPK1 inhibitor necrotstin-1 and by the heat shock protein 90 kDa inhibitor geldanamycin. Moreover both the staurosporine-triggered and the classical death ligand-induced necroptotic pathway can be effectively arrested by a lysosomal enzyme inhibitor CA-074-OMe and the recently discovered MLKL inhibitor necrosulfonamide. We also confirmed that the enzymatic role of poly(ADP-ribose)polymerase (PARP) is dispensable in necroptosis but it contributes to membrane disruption in secondary necrosis. In conclusion, we identified a novel way of necroptosis induction that can facilitate our understanding of the molecular mechanisms of necroptosis. Our results shed light on alternative application of staurosporine, as a possible anticancer therapeutic agent. Furthermore, we showed that the CA-074-OMe has a target in the signaling pathway leading to necroptosis. Finally, we could differentiate necroptotic and secondary necrotic processes based on participation of PARP enzyme.

Precision oncology is currently based on pairing molecularly targeted agents (MTA) to predefined single driver genes or biomarkers. Each tumor harbors a combination of a large number of potential genetic alterations of multiple driver genes in a complex system that limits the potential of this approach. We have developed an artificial intelligence (AI)-assisted computational method, the digital drug-assignment (DDA) system, to prioritize potential MTAs for each cancer patient based on the complex individual molecular profile of their tumor. We analyzed the clinical benefit of the DDA system on the molecular and clinical outcome data of patients treated in the SHIVA01 precision oncology clinical trial with MTAs matched to individual genetic alterations or biomarkers of their tumor. We found that the DDA score assigned to MTAs was significantly higher in patients experiencing disease control than in patients with progressive disease (1523 versus 580, P  = 0.037). The median PFS was also significantly longer in patients receiving MTAs with high (1000+ <) than with low (<0) DDA scores (3.95 versus 1.95 months, P  = 0.044). Our results indicate that AI-based systems, like DDA, are promising new tools for oncologists to improve the clinical benefit of precision oncology.

Several promising anti-cancer drug–GnRH (gonadotropin-releasing hormone) conjugates have been developed in the last two decades, although none of them have been approved for clinical use yet. Crizotinib is an effective multi-target kinase inhibitor, approved against anaplastic lymphoma kinase (ALK)- or ROS proto-oncogene 1 (ROS-1)-positive non-small cell lung carcinoma (NSCLC); however, its application is accompanied by serious side effects. In order to deliver crizotinib selectively into the tumor cells, we synthesized novel crizotinib analogues and conjugated them to a [d-Lys6]–GnRH-I targeting peptide. Our most prominent crizotinib–GnRH conjugates, the amide-bond-containing [d-Lys6(crizotinib*)]–GnRH-I and the ester-bond-containing [d-Lys6(MJ55*)]–GnRH-I, were able to bind to GnRH-receptor (GnRHR) and exert a potent c-Met kinase inhibitory effect. The efficacy of compounds was tested on the MET-amplified and GnRHR-expressing EBC-1 NSCLC cells. In vitro pharmacological profiling led to the conclusion that that crizotinib–GnRH conjugates are transported directly into lysosomes, where the membrane permeability of crizotinib is diminished. As a consequence of GnRHR-mediated endocytosis, GnRH-conjugated crizotinib bypasses its molecular targets—the ATP-binding site of RTKs— and is sequestered in the lysosomes. These results explained the lower efficacy of crizotinib–GnRH conjugates in EBC-1 cells, and led to the conclusion that drug escape from the lysosomes is a major challenge in the development of clinically relevant anti-cancer drug–GnRH conjugates.

The poor prognosis of head and neck squamous cell carcinoma (HNSCC) is partly due to the lack of reliable predictive markers. Connexin 43 (Cx43) protein and its cell-communication channels have been assigned tumor suppressor functions while the anti-apoptotic Bcl-2 (B-cell lymphoma-2) protein has been associated with negative prognostic significance in cancer. This study aimed to test the role of Cx43 protein on Bcl-2 expression, tumor progression and response to taxane-based treatment in HNSCC. Human papillomavirus (HPV) negative HNSCC cell lines were tested for paclitaxel sensitivity through measuring apoptosis induction, cell viability and changes in Cx43 and Bcl-2 levels using flow cytometry, cell viability assay, immunocytochemistry and western blot. Inhibition of Cx43 expression using siRNA increased Bcl-2 protein levels in SCC25 (tongue squamous cell carcinoma) cells, while forced Cx43 expression reduced Bcl-2 levels and supported paclitaxel cytotoxicity in FaDu (hypopharynx squamous cell carcinoma) cells. In vitro results were in line with protein expression and clinicopathological features tested in tissue microarray samples of HNSCC patients. Our data demonstrate that elevated Cx43 and reduced Bcl-2 levels may indicate HNSCC sensitivity to taxane-based treatments. On the contrary, silencing of the Cx43 gene GJA1 (gap junction protein alpha-1) can result in increased Bcl-2 expression and reduced paclitaxel efficiency. Clinical tumor-based analysis also confirmed the inverse correlation between Cx43 and Bcl-2 expression.

Efforts to repeat the success of pioneering molecularly targeted cancer drugs, such as trastuzumab, for particular patient populations have been hampered by factors such as a lack of correlation between the molecular markers used to select patients for treatment and the drug response. This article highlights lessons learned from the development of drugs targeting members of the epidermal growth factor receptor family, and discusses strategies to decrease the risk of failure in clinical trials by more effectively integrating molecular diagnostics into anticancer drug discovery and development. In the 1990s, the breast cancer drug trastuzumab (Herceptin; Genentech/Roche) — an antibody specific for human epidermal growth factor receptor 2 (HER2; also known as ERBB2) — was approved based on trials in which HER2 expression levels were used to select patients in clinical trials. This provided support for analogous efforts for drugs that target the epidermal growth factor receptor (EGFR). However, the development of these drugs, such as cetuximab (Erbitux; Bristol–Myers Squibb/Lilly) and gefitinib (Iressa; AstraZeneca), has revealed that EGFR expression is an insufficient and unreliable biomarker to select patients for EGFR-targeted therapies in both lung and colon cancer. Indeed, evidence on patient populations that are likely to respond to such therapies, on the basis of specific mutations in proteins of the targeted pathway, has only recently been clinically validated and incorporated into some of the drug labels. This article highlights lessons learned from the development of the first drugs targeting the EGFR family and discusses strategies to decrease the risk of failure in clinical development by more effectively integrating molecular diagnostics into anticancer drug discovery.

The incidence of head and neck squamous cell carcinomas is still growing, and the long-term prognosis of advanced disease remains poor. Only a fraction of head and neck cancers are sensitive to the EGFR-inhibitor cetuximab, which is the only registered targeted therapy available today. In several cancers, gene copy number alterations of MET and PIK3CA have been found to be prognostic and predictive for therapy response. The aim of this study was to systematically analyze in head and neck cancers the pathological characteristics and prognostic significance of copy number changes of MET and PIK3CA genes. MET and PIK3CA copy numbers were analyzed by fluorescence in situ hybridization in tumor samples of 152 patients. Expression of EGFR, p16, and Ki67 was studied by immunohistochemistry. High polysomy of PIK3CA (chromosome 3) was found in 20 % of cases and amplification in 4.5 %. Regarding MET, 35 % of cases showed low or high polysomy of the gene (chromosome 7), while no intra-chromosomal amplification of MET was detected. PIK3CA copy number gain (high polysomy or amplification) was significantly associated with shorter disease-specific survival, larger tumor volume, and lower p16 expression. MET copy number gain (low or high polysomy) in tumors was significantly associated with shorter disease-specific survival and lower level of EGFR. PIK3CA and MET may play an important role in oncogenesis of certain specific subtypes of head and neck cancer. There is an urgent need for the development of novel targeted therapies against these tumors associated with poor prognosis.

Background Rheumatoid Arthritis is a chronic disease leading to decreased quality of life with a rather variable response rate to Disease Modifying Anti Rheumatic Drugs. Methotrexate (MTX) is the gold standard therapy in Rheumatoid Arthritis. The Multidrug resistance Related Protein and Multi Drug Resistance protein 1, also called P-glycoprotein-170 transporters can alter the intracellular concentration of different drugs. Methotrexate is an MRP1 substrate and thus the functional activity of MRP1 might have a clinical impact on the efficiency of the Methotrexate-therapy in Rheumatoid Arthritis. Methods We have compared the functional Multidrug Activity Factors (MAF) of the MDR1 and MRP1 transporters of Peripheral Blood Leukocytes of 59 Rheumatoid Arthritis patients with various response rate to MTX-therapy (MTX-responder, MTX-resistant and MTX-intolerant RA-groups) and 47 non-RA controls in six different leukocyte subpopulations (neutrophil leukocytes, monocytes, lymphocytes, CD4+, CD8+ and CD19+ cells). There was a decreased MAF of RA patients compared to non- Rheumatoid Arthritis patients and healthy controls in the leukocyte subpopulations. There was a significant difference between the MAF values of the MTX-responder and MTX intolerant groups. But we have not found significant differences between the MAF values of the MTX-responder and MTX-resistant Rheumatoid Arthritis -groups. Results Our results suggest that MDR1 and MRP1 functional activity does not seem to affect the response rate to MTX-therapy of Rheumatoid Arthritis-patients, but it might be useful in predicting MTX-side effects. We have demonstrated the decreased functional MDR-activity on almost 60 Rheumatoid Arthritis patients, which can be interpreted as a sign of the immune-suppressive effect of the MTX-treatment.

Background: We present the case of a 50-year-old female whose metastatic pancreatic neuroendocrine tumor (pNET) diagnosis was delayed by the COVID-19 pandemic. The patient was in critical condition at the time of diagnosis due to the extensive tumor burden and failing liver functions. The clinical dilemma was to choose between two registered first-line molecularly-targeted agents (MTAs), sunitinib or everolimus, or to use chemotherapy to quickly reduce tumor burden. Methods: Cell-free DNA (cfDNA) from liquid biopsy was analyzed by next generation sequencing (NGS) using a comprehensive 591-gene panel. Next, a computational method, digital drug-assignment (DDA) was deployed for rapid clinical decision support. Results: NGS analysis identified 38 genetic alterations. DDA identified 6 potential drivers, 24 targets, and 79 MTAs. Everolimus was chosen for first-line therapy based on supporting molecular evidence and the highest DDA ranking among therapies registered in this tumor type. The patient’s general condition and liver functions rapidly improved, and CT control revealed partial response in the lymph nodes and stable disease elsewhere. Conclusion: Deployment of precision oncology using liquid biopsy, comprehensive molecular profiling, and DDA make personalized first-line therapy of advanced pNET feasible in clinical settings.

Background: The anaplastic lymphoma kinase (ALK) gene fusion rearrangement is a potent oncogene, accounting for 2–7% of lung adenocarcinomas, with higher incidence (17–20%) in non-smokers. ALK-positive tumors are sensitive to ALK tyrosine kinase inhibitors (TKIs), thus ALK-positive non-small-cell lung cancer (NSCLC) is currently spearheading precision medicine in thoracic oncology, with three generations of approved ALK inhibitors in clinical practice. However, these treatments are eventually met with resistance. At the molecular level, ALK-positive NSCLC is of the lowest tumor mutational burden, which possibly accounts for the high initial response to TKIs. Nevertheless, TP53 co-mutations are relatively frequent and are associated with adverse outcome of crizotinib treatment, whereas utility of next-generation ALK inhibitors in TP53-mutant tumors is still unknown. Methods: We report the case of an ALK-positive, TP53-mutant NSCLC patient with about five years survival on ALK TKIs with continued next-generation regimens upon progression. Results: The tumor showed progression on crizotinib, but long tumor control was achieved following the incremental administration of next-generation ALK inhibitors, despite lack of evident resistance mechanisms. Conclusion: TP53 status should be taken into consideration when selecting ALK-inhibitor treatment for personalized therapies. In TP53-mutant tumors, switching TKI generations may overcome treatment exhaustion even in the absence of ALK-dependent resistance mechanisms.