Multi-omics analysis identifies repurposing bortezomib in the treatment of kidney-, nervous system-, and hematological cancers.

Repurposing of FDA-approved drugs is a quick and cost-effective alternative to de novo drug development. Here, we identify genes involved in bortezomib sensitivity, predict cancer types that may benefit from treatment with bortezomib, and evaluate the mechanism-of-action of bortezomib in breast cancer (BT-474 and ZR-75-30), melanoma (A-375), and glioblastoma (A-172) cells in vitro. Cancer cell lines derived from cancers of the blood, kidney, nervous system, and skin were found to be significantly more sensitive to bortezomib than other organ systems. The in vitro studies confirmed that although bortezomib effectively inhibited the ß5 catalytic site in all four cell lines, cell cycle arrest was only induced in G2/M phase and apoptosis in A-375 and A-172 after 24h. The genomic and transcriptomic analyses identified 33 genes (e.g. ALDH18A1, ATAD2) associated with bortezomib resistance. Taken together, we identified biomarkers predictive of bortezomib sensitivity and cancer types that might benefit from treatment with bortezomib.

Integrated transcriptomics- and structure-based drug repositioning identifies drugs with proteasome inhibitor properties.

Computational pharmacogenomics can potentially identify new indications for already approved drugs and pinpoint compounds with similar mechanism-of-action. Here, we used an integrated drug repositioning approach based on transcriptomics data and structure-based virtual screening to identify compounds with gene signatures similar to three known proteasome inhibitors (PIs; bortezomib, MG-132, and MLN-2238). In vitro validation of candidate compounds was then performed to assess proteasomal proteolytic activity, accumulation of ubiquitinated proteins, cell viability, and drug-induced expression in A375 melanoma and MCF7 breast cancer cells. Using this approach, we identified six compounds with PI properties ((-)-kinetin-riboside, manumycin-A, puromycin dihydrochloride, resistomycin, tegaserod maleate, and thapsigargin). Although the docking scores pinpointed their ability to bind to the ß5 subunit, our in vitro study revealed that these compounds inhibited the ß1, ß2, and ß5 catalytic sites to some extent. As shown with bortezomib, only manumycin-A, puromycin dihydrochloride, and tegaserod maleate resulted in excessive accumulation of ubiquitinated proteins and elevated HMOX1 expression. Taken together, our integrated drug repositioning approach and subsequent in vitro validation studies identified six compounds demonstrating properties similar to proteasome inhibitors.

Tumor-infiltrating lymphocytes as a predictor of axillary and primary tumor pathological response after neoadjuvant chemotherapy in patients with breast cancer: a retrospective cohort study.

PURPOSE: Tumor-infiltrating lymphocytes (TILs) can predict complete pathological response (pCR) of tumor in the breast but not so well-defined in the axilla after neoadjuvant chemotherapy. Since axillary surgery is being increasingly de-escalated after NACT, we aimed to investigate the relationship between TILs and pCR in the axilla and breast, as well as survival amongst NACT patients. METHODS: Clinicopathological data on patients who underwent NACT between 2013 and 2020 were retrospectively examined. Specifically, pre-TILs (before NACT), post-TILs (after NACT) and ΔTIL (changes in TILs) were assessed. Primary endpoint was pCR and secondary endpoints were breast cancer-free interval (BCFI) and overall survival (OS). RESULTS: Two hundred and twenty patients with nodal metastases were included. Overall axillary and breast pCR rates were 42.7% (94/220) and 39.1% (86/220), respectively, whereas the combined pCR rate was 32.7% (72/220). High pre-TILs (OR 2.03, 95% CI 1.02-4.05; p = 0.04) predicted axillary pCR whereas, high post-TILs (OR 0.33, 95% CI 0.14-0.76; p = 0.009) and increased ΔTILs (OR 0.25, 95% CI 0.08-0.79; p = 0.02) predicted non-axillary pCR. TILs were not a significant predictor for BCFI and OS. CONCLUSIONS: This study supports the potential use of pre-TILs to select initially node-positive patients for axillary surgical de-escalation after NACT.

Repurposing proteasome inhibitors for improved treatment of triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is associated with poor prognosis and limited treatment options due to the lack of important receptors (estrogen receptor [ER], progesterone receptor [PR], and human epidermal growth factor receptor 2 [HER2]) used for targeted therapy. However, high-throughput in vitro drug screening of cell lines is a powerful tool for identifying effective drugs for a disease. Here, we determine the intrinsic chemosensitivity of TNBC cell lines to proteasome inhibitors (PIs), thereby identifying potentially potent 2-drug combinations for TNBC. Eight TNBC cell lines (BT-549, CAL-148, HCC1806, HCC38, HCC70, MDA-MB-436, MDA-MB-453, and MDA-MB-468) and two controls (MCF-10A and MCF-7) were first exposed to 18 drugs (11 PIs and 7 clinically relevant chemotherapeutic agents) as monotherapy, followed by prediction of potent 2-drug combinations using the IDACombo pipeline. The synergistic effects of the 2-drug combinations were evaluated with SynergyFinder in four TNBC cell lines (CAL-148, HCC1806, HCC38, and MDA-MB-468) and three controls (BT-474, MCF-7, and T47D) in vitro, followed by further evaluation of tumor regression in zebrafish tumor models established using HCC1806 and MCF-7 cells. Monotherapy identified nine effective drugs (bortezomib, carfilzomib, cisplatin, delanzomib, docetaxel, epoxomicin, MLN-2238, MLN-9708, and nedaplatin) across all cell lines. PIs (e.g., bortezomib, delanzomib, and epoxomicin) were highly potent drugs in TNBC cells, of which bortezomib and delanzomib inhibited the chymotrypsin-like activity of the 20 S proteasome by 100% at 10 µM. Moreover, several potent 2-drug combinations (e.g., bortezomib+nedaplatin and epoxomicin+epirubicin) that killed virtually 100% of cells were also identified. Although HCC1806- and MCF-7-derived xenografts treated with bortezomib+nedaplatin and carboplatin+paclitaxel were smaller, HCC1806 cells frequently metastasized to the trunk region. Taken together, we show that PIs used in combination with platinum agents or topoisomerase inhibitors exhibit increased efficiency with almost 100% inhibition in TNBC cell lines, indicating that PIs are therefore promising compounds to use as combination therapy for TNBC.

Patient-derived scaffolds representing breast cancer microenvironments influence chemotherapy responses in adapted cancer cells consistent with clinical features.

BACKGROUND: The tumor microenvironment clearly influences cancer progressing properties but less is known about how individual cancer microenvironments potentially moderate cancer treatment effects. By cultivating and treating cancer cell lines in patient-derived scaffolds (PDS), the impact of specific characteristics of individual cancer microenvironments can be incorporated in human-like growth modelling and cancer drug treatment testing. METHODS: PDSs from 78 biobanked primary breast cancer samples with known patient outcomes, were prepared and repopulated with donor breast cancer cell lines, followed by treatment with 5-fluorouracil or doxorubicin after cellular adaption to the various microenvironments. Cancer cell responses to the treatments were monitored by RNA-analyses, highlighting changes in gene sets representative for crucial tumor biological processes such as proliferation, cancer stem cell features, differentiation and epithelial-to-mesenchymal transition. RESULTS: The chemotherapy treatments induced distinct gene expression patterns in adapted cancer cells with clusters of similar treatment responses depending on the patient-derived cancer microenvironment used as growth substrate. The doxorubicin treatment displayed a favorable gene signature among surviving cancer cells with low proliferation (MKI67) and pluripotency features (NANOG, POU5F1), in comparison to 5-fluorouracil showing low proliferation but increased pluripotency. Specific gene changes monitored post-treatment were also significantly correlated with clinical data, including histological grade (NANOG), lymph node metastasis (SLUG) and disease-free patient survival (CD44). CONCLUSIONS: This laboratory-based treatment study using patient-derived scaffolds repopulated with cancer cell lines, clearly illustrates that the human cancer microenvironment influences chemotherapy responses. The differences in treatment responses defined by scaffold-cultures have potential prognostic and treatment predictive values.

SARS-CoV-2 Infection in Pemphigus Vulgaris Two Weeks after Rituximab Therapy with Total Recovery: A Case Report.

The mortality risk factors for Corona Virus Disease-19 (COVID-19) infection (caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2)) include advanced age, male sex, certain comorbidities, and immunosuppression (1). Pemphigus vulgaris is a rare mucocutaneous autoimmune disease with autoantibodies against desmosomal desmoglein-1 and desmoglein-3, resulting in acantholysis and blister formation. This epithelial barrier defect increases susceptibility to infections, which may lead to relapses (2). Additionally, therapy-associated immunosuppression can lead to severe infections. Corticosteroids are the mainstay therapy. For moderate and severe pemphigus, rituximab is recommended in first-line treatment along with other immunosuppressants, and it may also be added in refractory cases. It is a monoclonal antibody against CD20 with long-lasting B-cell depletion potency. Recovery of B-cell function may last from one to seven years. Consequently, patients receiving rituximab cannot produce enough COVID-19 specific plasma cells, leading to a severe course of COVID-19 (2). Shashidi-Dadras et al. reported five mild COVID-19 cases among 167 patients with pemphigus who had received rituximab one to five years earlier. The authors presumed rituximab use within five years increases COVID-19 susceptibility regardless the number of courses received (3). Among 48 patients with pemphigus treated with rituximab within five years, Uzuncakmak et al. reported one mild case of COVID-19 (in a patient who had received a single course seven months earlier) (4). In another study, high titers of SARS-CoV-2 antibodies and high counts of antibody-secreting cells were associated with severe COVID-19 (5), which may be the consequence of antibody-dependent enhancement (6). Mahmoudi et al. concluded that B-cells may not be necessary for recovery in COVID-19, but they may protect from reinfection (7). Considering these data, rituximab should be postponed during the pandemic (8). In exceptional cases, it may be applied with careful consideration of the risk-benefit ratio (2,4). Patients should be monitored for signs of COVID-19 before and during treatment. A 63-year-old woman with pemphigus vulgaris presented at our department with widespread skin lesions. Comorbidities included hypertension, hypothyroidism, and glaucoma. Diagnosis was established based on histology and direct and indirect immunofluorescent microscopy results. Both desmoglein-1 and desmoglein-3 autoantibodies were detectable by ELISA. The patient was initially treated with low-dose systemic methylprednisolone (8 mg/day), because glaucoma contraindicated a higher dose. Azathioprine was subsequently started (gradually increased from 0.6 to 2.5 mg/kg/day). Continuous mucocutaneous progression 4 weeks later led to the decision to add rituximab therapy. The patient was confirmed as SARS-CoV-2 negative and received 1000 mg 12 weeks after starting glucocorticoid treatment. Two weeks later, she developed fever and became SARS-CoV-2 positive, and therefore the second rituximab treatment had to be cancelled. The patient had fever for six weeks without any other complaints, hospitalization was not required, and immunosuppression was continued with 8 mg methylprednisolone and 2.5 mg/kg azathioprine. Two weeks after recovery, she was diagnosed with pulmonary embolism, but recovered completely. Pulmonary embolism is a relatively common complication of COVID-19 which may be triggered by inactivity, loss of body fluids due to fever, a hypercoagulable state, and direct toxic venous endothelial damage caused by the virus (9). At a follow-up 4 months later, minimal skin lesions and significantly decreased desmoglein-1 and desmoglein-3 titers were observed. Azathioprine and methylprednisolone therapy were continued, and a second dosage of rituximab was given 7 months from the first one without any side-effects. We conclude that rituximab is a highly effective therapy in pemphigus, but the risk-benefit ratio should be carefully considered during the COVID-19 pandemic. We have not observed irreversible or permanent consequences of its administration, but our patient had a potentially lethal complication, pulmonary embolism, which may be associated with a more severe COVID-19 course due to immunosuppression. Total recovery was observed despite COVID-19 shortly after the initiation of rituximab.