Piotr Zapala

B-cell receptor signaling in the pathogenesis of lymphoid malignancies

B-cell receptor (BCR) signaling pathway plays a central role in B-lymphocyte development and initiation of humoral immunity. Recently, BCR signaling pathway has been shown as a major driver in the pathogenesis of B-cell malignancies. As a result, a vast array of BCR-associated kinases has emerged as rational therapeutic targets changing treatment paradigms in B cell malignancies. Based on high efficacy in early-stage clinical trials, there is rapid clinical development of inhibitors targeting BCR signaling pathway. Here, we describe the essential components of BCR signaling, their function in normal and pathogenic signaling and molecular effects of their inhibition in vitro and in vivo.

Inhibitors of SRC kinases impair antitumor activity of anti-CD20 monoclonal antibodies

Clinical trials with SRC family kinases (SFKs) inhibitors used alone or in a combination with anti-CD20 monoclonal antibodies (mAbs) are currently underway in the treatment of B-cell tumors. However, molecular interactions between these therapeutics have not been studied so far. A transcriptional profiling of tumor cells incubated with SFKs inhibitors revealed strong downregulation of MS4A1 gene encoding CD20 antigen. In a panel of primary and established B-cell tumors we observed that SFKs inhibitors strongly affect CD20 expression at the transcriptional level, leading to inhibition of anti-CD20 mAbs binding and increased resistance of tumor cells to complement-dependent cytotoxicity. Activation of the AKT signaling pathway significantly protected cells from dasatinib-triggered CD20 downregulation. Additionally, SFKs inhibitors suppressed antibody-dependent cell-mediated cytotoxicity by direct inhibition of natural killer cells. Abrogation of antitumor activity of rituximab was also observed in vivo in a mouse model. Noteworthy, the effects of SFKs inhibitors on NK cell function are largely reversible. The results of our studies indicate that development of optimal combinations of novel treatment modalities with anti-CD20 mAbs should be preceded by detailed preclinical evaluation of their effects on target cells.

Safety and Efficacy of Intravesical Bacillus Calmette-Guérin Immunotherapy in Patients with Non-Muscle-Invasive Bladder Cancer Presenting with Asymptomatic Bacteriuria: A Systematic Review

Intravesical Bacillus Calmette-Guérin (BCG) immunotherapy in bladder cancer patients with asymptomatic bacteriuria (ABU) remains a matter of debate. The aim of this systematic review was to present available evidence on the safety and efficacy of BCG immunotherapy in patients with ABU. A literature search within the Medline and the Embase databases was conducted with the following search terms: adverse events, bacteriuria, BCG, bladder cancer, cystitis, infection, pyuria, side effects and urinary tract infection (UTI). Sixteen relevant original articles were identified, including 6 articles directly presenting the safety or efficacy of BCG therapy in patients with ABU. None of them was a randomized controlled trial. Intravesical BCG instillations in patients with ABU were not associated with the increased risk of symptomatic UTI and did not affect negatively the recurrence- or progression-free survival. Routine urine analysis before BCG instillation created increased cost and potentially unnecessary delays in BCG therapy. ABU does not affect negatively the safety and efficacy of intravesical BCG immunotherapy. There is no evidence to support routine screening and treatment of ABU in patients scheduled for intravesical BCG instillations due to bladder cancer. However, this issue was not addressed adequately and needs further research.

HDAC6 inhibition upregulates CD20 levels and increases the efficacy of anti-CD20 monoclonal antibodies

Downregulation of CD20, a molecular target for monoclonal antibodies (mAbs), is a clinical problem leading to decreased efficacy of anti-CD20-based therapeutic regimens. The epigenetic modulation of CD20 coding gene (MS4A1) has been proposed as a mechanism for the reduced therapeutic efficacy of anti-CD20 antibodies and confirmed with nonselective histone deacetylase inhibitors (HDACis). Because the use of pan-HDACis is associated with substantial adverse effects, the identification of particular HDAC isoforms involved in CD20 regulation seems to be of paramount importance. In this study, we demonstrate for the first time the role of HDAC6 in the regulation of CD20 levels. We show that inhibition of HDAC6 activity significantly increases CD20 levels in established B-cell tumor cell lines and primary malignant cells. Using pharmacologic and genetic approaches, we confirm that HDAC6 inhibition augments in vitro efficacy of anti-CD20 mAbs and improves survival of mice treated with rituximab. Mechanistically, we demonstrate that HDAC6 influences synthesis of CD20 protein independently of the regulation of MS4A1 transcription. We further demonstrate that translation of CD20 mRNA is significantly enhanced after HDAC6 inhibition, as shown by the increase of CD20 mRNA within the polysomal fraction, indicating a new role of HDAC6 in the posttranscriptional mechanism of CD20 regulation. Collectively, our findings suggest HDAC6 inhibition is a rational therapeutic strategy to be implemented in combination therapies with anti-CD20 monoclonal antibodies and open up novel avenues for the clinical use of HDAC6 inhibitors.

Clinical rationale and safety of restaging transurethral resection in indication-stratified patients with high-risk non-muscle-invasive bladder cancer

BackgroundIndications for restaging transurethral resection of the bladder tumor (reTURBT) in patients with non-muscle-invasive bladder cancer (NMIBC) remain controversial. This study was aimed at evaluation of clinical value and safety of reTURBT in different clinical indications.MethodsThis is a retrospective analysis of consecutive 141 patients who underwent TURBT followed by reTURBT in years 2011–2015 in a single department. Pathological results and surgical complications were analyzed in the whole study cohort and stratified by clinical stage (Ta, T1, Tx (no muscle in the specimen)) and grade (low-grade (LG), high-grade (HG)) of bladder cancer diagnosed at primary TURBT.ResultsFull data was available for 132 patients. Residual disease was found in 53 patients (40.2%) with highest rate for Ta-HG cases (57.1%) followed by T1-HG (51.4%), Tx-HG (45.2%), T1-LG (32.1%), and Tx-LG (25.8%). In the multivariate analysis, high grade (p = 0.02) was the only independent predictor of residual disease. Upstaging to muscle-invasive bladder cancer was noticed in 9 patients (6.8%). The rate of grade ≥ 2 Clavien-Dindo complications (1.5 vs. 5.3%) did not differ significantly between TURBT and reTURBT cases.ConclusionsReTURBT is a safe procedure that remains crucial for therapeutic and staging purposes in patients with T1, Tx, or high-grade bladder cancer found in the primary resection.

FOXO1 promotes resistance of Non-Hodgkin lymphomas to anti-CD20-based therapy

ABSTRACT Diminished overall survival rate of non-Hodgkin lymphoma (NHL) patients treated with a combination regimen of rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone (R-CHOP) has been recently linked to recurrent somatic mutations activating FOXO1. Despite of the clinical relevance of this finding, the molecular mechanism driving resistance to R-CHOP therapy remains largely unknown. Herein, we investigated the potential role of FOXO1 in the therapeutic efficacy of rituximab, the only targeted therapy included in the R-CHOP regimen. We found CD20 transcription is negatively regulated by FOXO1 in NHL cell lines and in human lymphoma specimens carrying activating mutations of FOXO1. Furthermore, both the expression of exogenous mutants of FOXO1 and the inhibition of AKT led to FOXO1 activation in lymphoma cells, increased binding to MS4A1 promoter and diminished CD20 expression levels. In contrast, a disruption of FOXO1 with CRISPR/Cas9 genome-editing (sgFOXO1) resulted in CD20 upregulation, improved the cytotoxicity induced by rituximab and the survival of mice with sgFOXO1 tumors. Accordingly, pharmacological inhibition of FOXO1 activity in primary samples upregulated surface CD20 levels. Importantly, FOXO1 was required for the downregulation of CD20 levels by the clinically tested inhibitors of BTK, SYK, PI3K and AKT. Taken together, these results indicate for the first time that the AKT-unresponsive mutants of FOXO1 are important determinant of cell response to rituximab-induced cytotoxicity, and suggest that the genetic status of FOXO1 together with its transcriptional activity need further attention while designing anti-CD20 antibodies based regimens for the therapy of pre-selected lymphomas.

What Can Be Expected from Prostate Cancer Biomarkers? A Clinical Perspective

Along with significant advances in prostate cancer biology research, we also observe the rapid development of modern diagnostic tests. New biomarkers are derived to detect disease while it is organ-confined to stratify the risk and to aid clinical decision-making. Majority of these tools have already been validated clinically, but only a few have received premarket clearance and administration approval. Superiority of novel tests is visible not only in improved detection accuracy but predominantly in the assessment of tumour aggressiveness and selection of patients eligible for conservative management. Two factors limiting the clinical implementation of validated biomarker candidates are costs and local availability. For these reasons, currently, their true clinical role starts after routine screening with prostate-specific antigen test. With this review of prostate cancer biomarkers, we attempted to draw general conclusions on clinical perspectives of these novel tools.

Abstract LB-242: PTEN regulates the CD20 antigen expression and affects rituximab-based therapy of lymphoma malignancies

The therapeutic strategies currently used in B cell malignancies include the treatment with monoclonal antibodies (rituximab and ofatumumab) directed against CD20 antigen. These antibodies specifically eliminate B cells by triggering indirect effector mechanisms of the immune system, like complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), or immunophagocytosis. In many patients, the reduced level of CD20 antigen on the surface of tumor B cells leads to the resistance to anti-CD20 therapy. The aim of this study was to explore the molecular mechanisms governing the regulation of CD20 expression in lymphoma cells as a potential explanation of the resistance to rituximab/ofatumumab therapy. We previously observed that CD20 mRNA expression is significantly affected by the SRC family inhibitors. Here, we report that also PTEN tumor suppressor is a negative regulator of CD20 expression. To uncover the transcriptional mechanisms governing the CD20 expression we employed the construct encoding the promoter region of CD20 cloned upstream of the firefly luciferase gene. Overexpression of wild-type PTEN (but not the phosphatase-deficient mutant) strongly affected the promoter activity and the expression of CD20, leading to decreased binding of rituximab and ofatumumab and increased resistance of tumor cells to complement-dependent cytotoxicity. Using the truncated versions of the CD20 promoter we identified a particular region (-313/-198) as the major region sensitive to PTEN overexpression. We found that the negative regulation of CD20 promoter activity by PTEN was mediated by inhibition of AKT signaling. We observed that the overexpression of constitutively active AKT1 (CA-AKT1) overcame the negative effect of PTEN and sensitized cells to rituximab/ofatumumab treatment. The results of our studies indicate that PTEN status in tumor cells should therefore be considered when analyzing the mechanisms of resistance of B cell malignancies to anti-CD20 therapies. Citation Format: Beata Pyrzynska, Kamil Bojarczuk, Marta Siernicka, Michal Dwojak, Malgorzata Bobrowicz, Nina Miazek, Piotr Zapala, Agnieszka Zagozdzon, Jakub Golab, Magdalena Winiarska. PTEN regulates the CD20 antigen expression and affects rituximab-based therapy of lymphoma malignancies. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-242. doi:10.1158/1538-7445.AM2015-LB-242