(A) Comparison of the extent of apopotosis induced by HHT (after 24hrs exposure) in HHT-resistant (HHT-R) clones compared to the original cell lines (CTRL). (B) Western blot analysis demonstrates upregulation of BCL-XL protein in HHT-R clones compared to CTRL cell lines.

Downregulation of MYC, cFLIP or BCL6 in OCI-Ly7 and BJAB DLBCL cell lines (by western blot) after exposure to HHT.

Western blot analysis showing protein expression profile of key regulators of apoptosis in 18 DLBCL cell lines (12 germinal center B-cell-like (GCB) and 6 activated B-cell-like (ABC) DLBCL cell lines). *UPF4D cell line (GCB origin) was derived in our laboratory.

Cell of origin (COO), BCL2 expression status and IC100 for ABT-199 (µM) and HHT (nM) are shown.

To investigate whether the expression level of BCL2 proteins detected by western blot is comparable with the expression level assessed by IHC analysis, we analyzed protein expression (by IHC) of BCL2, MCL1 and BCL-XL from subcutaneous lymphoma xenografts obtained from mice xenotransplanted with selected DLBCL cell lines with known protein expression (by western blot).

Viability of DLBCL cell lines cultured with different concentrations of HHT (nM) or ABT-199 (µM) was measured by WST-8 survival/proliferation assay for 7-14 days. Graphs indicate % of viable cells, i.e. % of maximal absorbance of untreated cells shown with dashed lines. Representative example of two independent experiments is shown. Standard deviations were < 5% for all measurements.

Shown are examples of different levels of semi-quantitative protein expression (0-3).

The graphs show the extent of apoptosis of DLBCL cell lines induced by different concentrations of HHT, ABT-199 and ABT-737 after 24hrs exposure to particular agents.

Supplemental Figure 1. Next-generation sequencing of PDXs and primary MCL cells Supplemental Figure 2. IHC analysis of primary MCL samples and murine xenografts Supplemental Figure 3. Array comparative genomic hybridization of 24 primary MCL samples Supplemental Figure 4. Sensitivity of HBL2 and MAVER-1 cells resistant to venetoclax to BCL-XL inhibitors WEHI-539 and A1155463 Supplemental Table 1. Complete list of protein coding variants Supplemental Table 2. FISH analyses of the established murine PDXs of MCL Supplemental Table 3. Baseline characteristics of patients Supplemental Table 4. IHC analysis of MCL samples and xenografts Supplemental Table 5. Tumor and spleen weights at the end of in vivo experiments

218 Main document: 4303 (Introduction, Methods, Results, Discussion) Figures: 5 Tables: 1 Supplemental data for review: Supplemental Figures: 4 Supplemental Tables: 5 Translational Relevance: BCL2-targeting agent venetoclax (VTX) has promising anticancer activity in mantle cell lymphoma (MCL), but remissions tend to be short, which calls for rational drug combinations. We demonstrated that MCL1 and NOXA play important roles in mediating resistance to VTX. Consequently, we proposed an experimental treatment strategy based on co-targeting BCL2 with VTX and MCL1 with a highly specific small molecule MCL1 inhibitor S63845,. The combination of VTX and S63845 demonstrated synthetic lethality in vivo on a panel of five patient-derived xenografts established from patients with relapsed MCL with adverse cytogenetics. Research. on May 2, 2021. © 2019 American Association for Cancer clincancerres.aacrjournals.org Downloaded from Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on April 19, 2019; DOI: 10.1158/1078-0432.CCR-18-3275

Abstract Purpose: Mantle cell lymphoma (MCL) is an aggressive subtype of B-cell non-Hodgkin lymphomas characterized by (over)expression of BCL2. A BCL2-targeting drug, venetoclax, has promising anticancer activity in MCL. We analyzed molecular mechanisms of venetoclax resistance in MCL cells and tested strategies to overcome it. Experimental Design: We confirmed key roles of proapoptotic proteins BIM and NOXA in mediating venetoclax-induced cell death in MCL. Both BIM and NOXA are, however, differentially expressed in cell lines compared with primary cells. First, NOXA protein is significantly overexpressed in most MCL cell lines. Second, deletions of BIM gene harbored by three commonly used MCL cell lines (JEKO-1, MINO, and Z138) were not found by array comparative genomic hybridization using a validation set of 24 primary MCL samples. Results: We demonstrated that MCL1 and NOXA play important roles in mediating resistance to venetoclax. Consequently, we tested an experimental treatment strategy based on cotargeting BCL2 with venetoclax and MCL1 with a highly specific small-molecule MCL1 inhibitor S63845. The combination of venetoclax and S63845 demonstrated synthetic lethality in vivo on a panel of five patient-derived xenografts established from patients with relapsed MCL with adverse cytogenetics. Conclusions: Our data strongly support investigation of venetoclax in combination with S63845 as an innovative treatment strategy for chemoresistant MCL patients with adverse cytogenetics in the clinical grounds.

Richter syndrome represents the transformation of the chronic lymphocytic leukemia (CLL) into an aggressive lymphoma, most frequently the diffuse large B‐cell lymphoma (DLBCL). In this report we describe a patient with CLL, who developed a clonally‐related pleomorphic highly‐aggressive mantle cell lymphoma (MCL) after five cycles of a fludarabine‐based second‐line therapy for the first relapse of CLL. Molecular cytogenetic methods together with whole‐exome sequencing revealed numerous gene alterations restricted to the MCL clone (apart from the canonical t(11;14)(q13;q32) translocation) including gain of one copy of ATM gene or emergence of TP53 , CREBBP , NUP214 , FUBP1 and SF3B1 gene mutations. Similarly, gene expression analysis revealed vast differences between the MCL and CLL transcriptome, including overexpression of cyclin D1, downregulation of cyclins D2 and D3, or downregulation of IL4R in the MCL clone. Backtracking analysis using quantitative PCR specifically detecting an MCL‐restricted focal deletion of TP53 revealed that the pre‐MCL clone appeared in the bone marrow and peripheral blood of the patient approximately 4 years before the clinical manifestation of MCL. Both molecular cytogenetic and sequencing data support the hypothesis of a slow development of the pre‐MCL clone in parallel to CLL over several years, and thereby exclude the possibility that the transformation event occurred at the stage of the CLL relapse clone by mere t(11;14)(q13;q32) acquisition.

Abstract Purpose: To investigate the roles of BCL2, MCL1, and BCL-XL in the survival of diffuse large B-cell lymphoma (DLBCL). Experimental designs: Immunohistochemical analysis of 105 primary DLBCL samples, and Western blot analysis of 18 DLBCL cell lines for the expression of BCL2, MCL1, and BCL-XL. Pharmacologic targeting of BCL2, MCL1, and BCL-XL with ABT-199, homoharringtonine (HHT), and ABT-737. Analysis of DLBCL clones with manipulated expressions of BCL2, MCL1, and BCL-XL. Immunoprecipitation of MCL1 complexes in selected DLBCL cell lines. Experimental therapy aimed at inhibition of BCL2 and MCL1 using ABT-199 and HHT, single agent, or in combination, in vitro and in vivo on primary cell-based murine xenograft models of DLBCL. Results: By the pharmacologic targeting of BCL2, MCL1, and BCL-XL, we demonstrated that DLBCL can be divided into BCL2-dependent and MCL1-dependent subgroups with a less pronounced role left for BCL-XL. Derived DLBCL clones with manipulated expressions of BCL2, MCL1, and BCL-XL, as well as the immunoprecipitation experiments, which analyzed MCL1 protein complexes, confirmed these findings at the molecular level. We demonstrated that concurrent inhibition of BCL2 and MCL1 with ABT-199 and HHT induced significant synthetic lethality in most BCL2-expressing DLBCL cell lines. The marked cytotoxic synergy between ABT-199 and HHT was also confirmed in vivo using primary cell-based murine xenograft models of DLBCL. Conclusions: As homoharringtonine is a clinically approved antileukemia drug, and ABT-199 is in advanced phases of diverse clinical trials, our data might have direct implications for novel concepts of early clinical trials in patients with aggressive DLBCL. Clin Cancer Res; 22(5); 1138–49. ©2015 AACR.

Complex laboratory investigation is necessary for the diagnosis and relevant classification of lymphomas. The classical histopathological morphology and cytology investigation is essential, but further investigations such as immunohistochemistry and fluorescence in situ hybridization are necessary. It is also important to employ flow cytometry as a method of investigation running synchronously or preceding the histopathological approach. Last but not least, the investigation of nucleic acids in lymphoma by molecular approaches is necessary and has become an everyday practice. Communication between pathologists and clinical colleagues (oncologists, hematologists, internal medicine specialists and radiologists) is very important. We demonstrate the necessity of a complex diagnostic approach to lymphomas and an appropriate interpretation of all laboratory investigations giving examples of eight patients with various types of lymphomas. In some cases, it is impossible to properly diagnose a lymphoma without molecular investigation. Occasionally, the results of the molecular investigation may be misleading and/or may be inaccurately interpreted, leading to an incorrect conclusion. For that reason, it is very important to incorporate all specialized laboratories and their teams under one roof (preferably that of pathology departments), enabling tight and daily cooperation between the specialists. This is the way to reach a precise diagnosis in a majority of cases, as well as how to comply with clinical expectations of properly classified lymphomas for a targeted therapy of patients.

The formation and preservation of sandstone landforms such as pillars and arches is enigmatic. Experiments and numerical modelling show that load-bearing material weathers more slowly, and thus the internal stress field can shape and stabilize sandstone landforms.

Abstract Introduction: Diffuse large B-cell lymphoma (DLBCL) represents the most prevalent type of B-cell non-Hodgkin lymphomas (B-NHL) in the Western hemisphere. While BCL2 gene deregulation was repeatedly associated with poor prognosis, the role of MCL1 in the biology of DLBCL remains largely unknown. ABT199 is a highly-selective inhibitor of BCL2 protein currently evaluated in clinical trials. Homoharringtonine (HHT) is a plant alkaloid and as a semisynthetic compound (omacetaxine) it was approved for the treatment of relapsed chronic myelogenous leukemia (CML). Anti-tumor activity of HHT includes downregulation of the anti-apoptotic protein MCL1. Aim: The aim of the project was to evaluate the preclinical anti-lymphoma efficacy of BCL2 and MCL1-targeting agents ABT199 and HHT in DLBCL. Methods: Immunophenotype of primary DLBCL samples was determined by immunohistochemistry (IHC) using the Hans algorithm. Sensitivity of DLBCL cell lines to ABT199 and HHT was determined by Annexin V-based apoptotic assay and WST8-based cell proliferation assay. DLBCL clones with downregulation of selected anti-apoptotic proteins were derived using pLKO1-based lentiviral particles containing shRNAs against BCL2, BCL-XL and MCL1. For upregulation, BCL2, BCL-XL and MCL1 were cloned in the lentiviral expression vector pCDHNeo and the prepared lentiviral particles were used for the transduction of DLBCL cell lines. Results: We analyzed molecular mechanisms of cytotoxic activity of HHT in 7 DLBCL cell lines, and confirmed decreased expression of MCL1 protein in all cases. By semi-quantitative protein expression analysis (western blot or IHC) we demonstrated that BCL-XL and MCL1 were detectable in all DLBCL cell lines (n=18) and primary samples (n=114, GCB=51, ABC=63), while BCL2 was not detectable in 6 out of 18 DLBCL cell lines and 32 out of 114 primary DLBCL samples. 8 out of 12 BCL2-positive DLBCL cell lines were sensitive to 1 microM ABT199 (i.e. did not survive 1 microM ABT199 by standard proliferation assay). In contrary, 6 out of 6 BCL2-negative DLBCL cell lines were resistant to 1 microM ABT199. 11 out of 12 BCL2-positive DLBCL cell lines were sensitive to 30 nM HHT (considered a steady-state plasma level in CML patients treated with HHT). 5 out of 6 BCL2-negative DLBCL cell lines were sensitive to 30 nM HHT. Significant drug synergism between ≤1 microM ABT199 and ≤ 30 nM HHT was observed in 8 out of 12 BCL2-positive, but only in 1 out of 6 BCL2-negative DLBCL cell lines. We demonstrated that high expression of BCL2 positively correlated with sensitivity to ABT-199, irrespective of expression levels of BCL-XL and MCL1. Expression levels of BCL2 and BCL-XL negatively correlated with sensitivity to HHT. Expression level of MCL1 did not correlate with sensitivity to HHT. Both targeted downregulation and transgenic overexpression of BCL-XL in selected DLBCL cell lines confirmed that the expression of BCL-XL negatively correlates with sensitivity to HHT (but not to ABT199). While increase in sensitivity to HHT was observed in 3 out of 3 DLBCL cell lines with targeted knock-down of BCL2, increase in sensitivity to ABT199 was observed only in 1 out of these 3 DLBCL cell lines. Targeted knockdown of MCL1 was associated with increased sensitivity to HHT in 1 out of 2 DLBCL cell lines, but with no change of sensitivity to ABT199. Conclusions: HHT is a promising anti-DLBCL agent in both BCL2-positive and BCL2-negative cases. ABT199, as a single-agent or in combination with HHT, effectively eliminates BCL2-positive DLBCL cells. Based on the observed data two biological categories of DLBCL might be assumed: BCL2-dependent (ABT199-sensitive, HHT-sensitive) and MCL1-dependent (ABT199-resistant, HHT-sensitive) DLBCL. Grant support: IGA-MZ: NT13201-4/2012, GACR14-19590S, UNCE 204021, SVV-2013-266509, PRVOUK P24/LF1/3, GA-UK 1270214 Disclosures No relevant conflicts of interest to declare.

In Strelec Quarry, the Czech Republic, an underground conduit network >300 m long with a volume of similar to 10(4) m(3) and a catchment of 7 km(2) developed over 5 years by groundwater flow in Cretaceous marine quartz sandstone. Similar landforms at natural exposures (conduits, slot canyons, undercuts) are stabilized by case hardening and have stopped evolving. The quarry offers a unique opportunity to study conduit evolution in sandstone at local to regional scales, from the initial stage to maturity, and to characterize the erosion processes which may form natural landforms prior to stabilization. A new technique was developed to distinguish erodible and non-erodible sandstone surfaces. Based on measurements of relative erodibility, drilling resistance, ambient and water-saturated tensile strength (TS) at natural and quarry exposures three distinct kinds of surfaces were found. 1) Erodible sandstone exposed at similar to 60% of surfaces in quarry. This sandstone loses as much as 99% of TS when saturated. 2) Sub-vertical fracture surfaces that are non-erodible already prior to exposure at ground surface and which keep considerable TS if saturated. 3) Case hardened surfaces that start to form after exposure. In favorable conditions they became non-erodible and reach the full TS in just 6 years. An increase in the hydraulic gradient from similar to 0.005 to >0.02 triggered conduit evolution, based on long-term monitoring of water table in 18 wells and inflows to the quarry. Rapidly evolving major conduits are characterized by a channel gradient of similar to 0.01, a flow velocity similar to 40 cm/s and sediment concentration similar to 10 g/l. Flow in openings with a discharge 1 ml/s and hydraulic gradient >0.05 exceeds the erosion threshold and initiates piping. In the first phase of conduit evolution, fast concentrated flow mobilizes erodible sandstone between sets of parallel fractures in the shallow phreatic zone. In the second phase the conduit opening mainly expands vertically upward into the vadose zone by mass wasting of undercut sandstone slabs. Mass wasting is responsible for >90% of mobilized sandstone. Sides of the mature conduits are protected by non-erodible fracture surfaces.Natural landforms were probably formed very rapidly by overland flow, piping and possibly fluidization during or at the end of the glacial periods when sandstone was not yet protected by case hardening. (c) 2012 Elsevier B.V. All rights reserved.