ORCID Profile
0000-0002-4493-306X
Current Organisation
Amsterdam UMC Locatie VUmc
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Publisher: American Society of Hematology
Date: 17-11-2022
Abstract: Venetoclax (VEN) inhibits the prosurvival protein BCL2 to induce apoptosis and is a standard therapy for chronic lymphocytic leukemia (CLL), delivering high complete remission rates and prolonged progression-free survival in relapsed CLL but with eventual loss of efficacy. A spectrum of subclonal genetic changes associated with VEN resistance has now been described. To fully understand clinical resistance to VEN, we combined single-cell short- and long-read RNA-sequencing to reveal the previously unappreciated scale of genetic and epigenetic changes underpinning acquired VEN resistance. These appear to be multilayered. One layer comprises changes in the BCL2 family of apoptosis regulators, especially the prosurvival family members. This includes previously described mutations in BCL2 and lification of the MCL1 gene but is heterogeneous across and within in idual patient leukemias. Changes in the proapoptotic genes are notably uncommon, except for single cases with subclonal losses of BAX or NOXA. Much more prominent was universal MCL1 gene upregulation. This was driven by an overlying layer of emergent NF-κB (nuclear factor kappa B) activation, which persisted in circulating cells during VEN therapy. We discovered that MCL1 could be a direct transcriptional target of NF-κB. Both the switch to alternative prosurvival factors and NF-κB activation largely dissipate following VEN discontinuation. Our studies reveal the extent of plasticity of CLL cells in their ability to evade VEN-induced apoptosis. Importantly, these findings pinpoint new approaches to circumvent VEN resistance and provide a specific biological justification for the strategy of VEN discontinuation once a maximal response is achieved rather than maintaining long-term selective pressure with the drug.
Publisher: Springer Science and Business Media LLC
Date: 25-01-2021
DOI: 10.1038/S41418-020-00692-W
Abstract: In chronic lymphocytic leukemia (CLL), the lymph node (LN) microenvironment delivers critical survival signals by inducing the expression of anti-apoptotic Bcl-2 members Bcl-XL, Bfl-1, and Mcl-1, resulting in apoptosis blockade. We determined previously that resistance against various drugs, among which is the clinically applied BH3 mimetic venetoclax, is dominated by upregulation of the anti-apoptotic regulator Bcl-XL. Direct clinical targeting of Bcl-XL by, e.g., Navitoclax is however not desirable due to induction of thrombocytopenia. Since the actual regulation of Bcl-XL in CLL in the context of the LN microenvironment is not well elucidated, we investigated various candidate LN signals to drive Bcl-XL expression. We found a dominance for NF-κB signaling upon CD40 stimulation, which results in activation of both the canonical and non-canonical NF-κB signaling pathways. We demonstrate that expression of Bcl-XL is first induced by the canonical NF-κB pathway, and subsequently boosted and continued via non-canonical NF-κB signaling through stabilization of NIK. NF-κB subunits p65 and p52 can both bind to the Bcl-XL promoter and activate transcription upon CD40 stimulation. Moreover, canonical NF-κB signaling was correlated with Bfl-1 expression, whereas Mcl-1 in contrast, was not transcriptionally regulated by NF-κB. Finally, we applied a novel compound targeting NIK to selectively inhibit the non-canonical NF-κB pathway and showed that venetoclax-resistant CLL cells were sensitized to venetoclax. In conclusion, protective signals from the CLL microenvironment can be tipped towards apoptosis sensitivity by interfering with non-canonical NF-κB signaling.
Publisher: Cold Spring Harbor Laboratory
Date: 08-11-2021
DOI: 10.1101/2021.11.06.467575
Abstract: Despite numerous methodological advances, the normalization of single cell RNA-seq (scRNA-seq) data remains a challenging task and the performance of different methods can vary greatly across datasets. Part of the reason for this is the different kinds of unwanted variation, including library size, batch and cell cycle effects, and the association of these with the biology embodied in the cells. A normalization method that does not explicitly take into account cell biology risks removing some of the signal of interest. Furthermore, most normalization methods remove the effects of unwanted variation for the cell embedding used for clustering-based analysis but not from gene-level data typically used for differential expression (DE) analysis to identify marker genes. Here we propose RUV-III-NB, a statistical method that can be used to remove unwanted variation from both the cell embedding and gene-level counts. RUV-III-NB explicitly takes into account its potential association with biology when removing unwanted variation via the use of pseudo-replicates. The method can be used for both UMI or sequence read counts and returns adjusted counts that can be used for downstream analyses such as clustering, DE and pseudotime analyses. Using five publicly available datasets that encompass different technological platforms, kinds of biology and levels of association between biology and unwanted variation, we show that RUV-III-NB manages to remove library size and batch effects, strengthen biological signals, improve differential expression analyses, and lead to results exhibiting greater concordance with independent datasets of the same kind. The performance of RUV-III-NB is consistent across the five datasets and is not sensitive to the number of factors assumed to contribute to the unwanted variation. It also shows promise for removing other kinds of unwanted variation such as platform effects. The method is implemented as a publicly available R package available from imfuxing/ruvIIInb .
Publisher: Elsevier BV
Date: 10-2019
DOI: 10.1016/J.CCELL.2019.09.008
Abstract: In this issue of Cancer Cell, Guièza et al. describe that overexpression of the pro-survival protein MCL1 and cellular energy metabolic reprogramming can contribute to resistance to the BCL2 inhibitor venetoclax in patients with chronic lymphocytic leukemia. This adds a new dimension to understanding of secondary clinical resistance to venetoclax.
Publisher: Springer Science and Business Media LLC
Date: 02-05-2017
DOI: 10.1038/LEU.2017.129
Abstract: The clinical success of B-cell receptor (BCR) signaling pathway inhibitors in chronic lymphocytic leukemia (CLL) is attributed to inhibition of adhesion in and migration towards the lymph node. Proliferation of CLL cells is restricted to this protective niche, but the underlying mechanism(s) is/are not known. Treatment with BCR pathway inhibitors results in rapid reductions of total clone size, while CLL cell survival is not affected, which points towards inhibition of proliferation. In vitro, BCR stimulation does not induce proliferation of CLL, but triggering via Toll-like receptor, tumor necrosis factor or cytokine receptors does. Here, we investigated the effects of clinically applied inhibitors that target BCR signaling, in the context of proliferation triggered either via CD40L/IL-21 or after CpG stimulation. CD40L/IL-21-induced proliferation could be inhibited by idelalisib and ibrutinib. We demonstrate this was due to blockade of CD40L-induced ERK-signaling. Targeting JAKs, but not SYK, blocked CD40L/IL-21-induced proliferation. In contrast, PI3K, BTK as well as SYK inhibition prevented CpG-induced proliferation. Knockdown experiments showed that CD40L/IL-21 did not co-opt upstream BCR components such as CD79A, in contrast to CpG-induced proliferation. Our data indicate that currently applied BTK/PI3K inhibitors target antigen-independent proliferation in CLL, and suggest that targeting of JAK and/or SYK might be clinically useful.
Publisher: Springer Science and Business Media LLC
Date: 18-06-2020
Publisher: Springer Science and Business Media LLC
Date: 19-11-2018
DOI: 10.1038/S41591-018-0243-Z
Abstract: Ibrutinib plus venetoclax is a highly effective combination in mantle cell lymphoma. However, strategies to enable the evaluation of therapeutic response are required. Our prospective analyses of patients within the AIM study revealed genomic profiles that clearly dichotomized responders and nonresponders. Mutations in ATM were present in most patients who achieved a complete response, while chromosome 9p21.1-p24.3 loss and/or mutations in components of the SWI-SNF chromatin-remodeling complex were present in all patients with primary resistance and two-thirds of patients with relapsed disease. Circulating tumor DNA analysis revealed that these alterations could be dynamically monitored, providing concurrent information on treatment response and tumor evolution. Functional modeling demonstrated that compromise of the SWI-SNF complex facilitated transcriptional upregulation of BCL2L1 (Bcl-xL) providing a selective advantage against ibrutinib plus venetoclax. Together these data highlight important insights into the molecular basis of therapeutic response and provide a model for real-time assessment of innovative targeted therapies.
Publisher: American Society of Hematology
Date: 24-10-2013
DOI: 10.1182/BLOOD-2012-11-467670
Abstract: Autologous activated T cells can drive antigen-independent proliferation of CLL cells through CD40 and IL-21 signaling. An IL-21 gene induction signature, IL-21 mRNA, and protein can be found in CLL lymph node s les.
Publisher: American Society of Hematology
Date: 05-03-2020
Abstract: The BCL2 inhibitor venetoclax has complete response rates of up to 50% in chronic lymphocytic leukemia patients, but secondary resistance reflecting acquired mutations in BCL2 can lead to treatment failure. Blombery et al report that an unexpectedly large number of patients carry multiple BCL2 mutations with subclonal variation in their occurrence.
Publisher: Springer Science and Business Media LLC
Date: 03-06-2019
DOI: 10.1038/S41467-019-10363-1
Abstract: Venetoclax is a first-in-class cancer therapy that interacts with the cellular apoptotic machinery promoting apoptosis. Treatment of patients suffering chronic lymphocytic leukaemia with this BCL-2 antagonist has revealed emergence of a drug-selected BCL-2 mutation (G101V) in some patients failing therapy. To understand the molecular basis of this acquired resistance we describe the crystal structures of venetoclax bound to both BCL-2 and the G101V mutant. The pose of venetoclax in its binding site on BCL-2 reveals small but unexpected differences as compared to published structures of complexes with venetoclax analogues. The G101V mutant complex structure and mutant binding assays reveal that resistance is acquired by a knock-on effect of V101 on an adjacent residue, E152, with venetoclax binding restored by a E152A mutation. This provides a framework for considering analogues of venetoclax that might be effective in combating this mutation.
Publisher: Springer Science and Business Media LLC
Date: 26-11-2019
DOI: 10.1038/S41388-019-1122-X
Abstract: Apoptosis-regulating BCL-2 family members, which can promote malignant transformation and resistance to therapy, have become prime therapeutic targets, as illustrated by the striking efficacy in certain lymphoid malignancies of the BCL-2-specific inhibitor venetoclax. In other lymphoid malignancies, however, such as the aggressive mantle cell lymphoma (MCL), cell survival might rely instead or also on BCL-2 relative MCL-1. We have explored MCL-1 as a target for killing MCL cells by both genetic and pharmacologic approaches. In several MCL cell lines, MCL-1 knockout with an inducible CRISPR/Cas9 system triggered spontaneous apoptosis. Accordingly, most MCL cell lines proved sensitive to the specific MCL-1 inhibitor S63845, and MCL-1 inhibition also proved efficacious in an MCL xenograft model. Furthermore, its killing efficacy rose on combination with venetoclax, the BCL-X
Publisher: American Society of Hematology
Date: 12-03-2020
Abstract: The BCL-2 inhibitor venetoclax combined with hypomethylating agents or low-dose cytarabine represents an important new therapy for older or unfit patients with acute myeloid leukemia (AML). We analyzed 81 patients receiving these venetoclax-based combinations to identify molecular correlates of durable remission, response followed by relapse (adaptive resistance), or refractory disease (primary resistance). High response rates and durable remissions were typically associated with NPM1 or IDH2 mutations, with prolonged molecular remissions prevalent for NPM1 mutations. Primary and adaptive resistance to venetoclax-based combinations was most commonly characterized by acquisition or enrichment of clones activating signaling pathways such as FLT3 or RAS or biallelically perturbing TP53. Single-cell studies highlighted the polyclonal nature of intratumoral resistance mechanisms in some cases. Among cases that were primary refractory, we identified heterogeneous and sometimes ergent interval changes in leukemic clones within a single cycle of therapy, highlighting the dynamic and rapid occurrence of therapeutic selection in AML. In functional studies, FLT3 internal tandem duplication gain or TP53 loss conferred cross-resistance to both venetoclax and cytotoxic-based therapies. Collectively, we highlight molecular determinants of outcome with clinical relevance to patients with AML receiving venetoclax-based combination therapies.
Publisher: American Society of Hematology
Date: 28-07-2016
DOI: 10.1182/BLOOD-2016-02-700328
Abstract: TORK/DNA-PK inhibition induces cytotoxicity and blocks signaling pathways important for CLL survival, proliferation, and drug resistance. Preliminary clinical effects of TORK/DNA-PK inhibition show 7 of 8 CLL patients with decreased lymphadenopathy.
Publisher: Cold Spring Harbor Laboratory
Date: 10-08-2020
DOI: 10.1101/2020.08.10.243543
Abstract: Alternative splicing shapes the phenotype of cells in development and disease. Long-read RNA-sequencing recovers full-length transcripts but has limited throughput at the single-cell level. Here we developed single-cell full-length transcript sequencing by s ling ( FLT-seq ), together with the computational pipeline FLAMES to overcome these issues and perform isoform discovery and quantification, splicing analysis and mutation detection in single cells. With FLT-seq and FLAMES , we performed the first comprehensive characterization of the full-length isoform landscape in single cells of different types and species and identified thousands of unannotated isoforms. We found conserved functional modules that were enriched for alternative transcript usage in different cell populations, including ribosome biogenesis and mRNA splicing. Analysis at the transcript-level allowed data integration with scATAC-seq on in idual promoters, improved correlation with protein expression data and linked mutations known to confer drug resistance to transcriptome heterogeneity. Our methods reveal previously unseen isoform complexity and provide a better framework for multi-omics data integration.
Publisher: American Association for Cancer Research (AACR)
Date: 03-2019
DOI: 10.1158/2159-8290.CD-18-1119
Abstract: Why CLL recurs in patients who achieve remission with the BCL2 inhibitor venetoclax has been unknown. We provide the first description of an acquired point mutation in BCL2 arising recurrently and exclusively in venetoclax-treated patients. The mutation reduces venetoclax binding and is sufficient to confer resistance. See related commentary by Thangava el and Byrd, p. 320. This article is highlighted in the In This Issue feature, p. 305
Publisher: Ferrata Storti Foundation (Haematologica)
Date: 08-05-2015
Publisher: Oxford University Press (OUP)
Date: 27-06-2022
DOI: 10.1093/NAR/GKAC486
Abstract: Normalization of single cell RNA-seq data remains a challenging task. The performance of different methods can vary greatly between datasets when unwanted factors and biology are associated. Most normalization methods also only remove the effects of unwanted variation for the cell embedding but not from gene-level data typically used for differential expression (DE) analysis to identify marker genes. We propose RUV-III-NB, a method that can be used to remove unwanted variation from both the cell embedding and gene-level counts. Using pseudo-replicates, RUV-III-NB explicitly takes into account potential association with biology when removing unwanted variation. The method can be used for both UMI or read counts and returns adjusted counts that can be used for downstream analyses such as clustering, DE and pseudotime analyses. Using published datasets with different technological platforms, kinds of biology and levels of association between biology and unwanted variation, we show that RUV-III-NB manages to remove library size and batch effects, strengthen biological signals, improve DE analyses, and lead to results exhibiting greater concordance with independent datasets of the same kind. The performance of RUV-III-NB is consistent and is not sensitive to the number of factors assumed to contribute to the unwanted variation.
Publisher: Springer Science and Business Media LLC
Date: 11-11-2021
DOI: 10.1186/S13059-021-02525-6
Abstract: A modified Chromium 10x droplet-based protocol that subs les cells for both short-read and long-read (nanopore) sequencing together with a new computational pipeline ( FLAMES ) is developed to enable isoform discovery, splicing analysis, and mutation detection in single cells. We identify thousands of unannotated isoforms and find conserved functional modules that are enriched for alternative transcript usage in different cell types and species, including ribosome biogenesis and mRNA splicing. Analysis at the transcript level allows data integration with scATAC-seq on in idual promoters, improved correlation with protein expression data, and linked mutations known to confer drug resistance to transcriptome heterogeneity.
Publisher: American Society of Hematology
Date: 20-05-2021
Abstract: Selective targeting of BCL-2 with the BH3-mimetic venetoclax has been a transformative treatment for patients with various leukemias. TP-53 controls apoptosis upstream of where BCL-2 and its prosurvival relatives, such as MCL-1, act. Therefore, targeting these prosurvival proteins could trigger apoptosis across erse blood cancers, irrespective of TP53 mutation status. Indeed, targeting BCL-2 has produced clinically relevant responses in blood cancers with aberrant TP-53. However, in our study, TP53-mutated or -deficient myeloid and lymphoid leukemias outcompeted isogenic controls with intact TP-53, unless sufficient concentrations of BH3-mimetics targeting BCL-2 or MCL-1 were applied. Strikingly, tumor cells with TP-53 dysfunction escaped and thrived over time if inhibition of BCL-2 or MCL-1 was sublethal, in part because of an increased threshold for BAX/BAK activation in these cells. Our study revealed the key role of TP-53 in shaping long-term responses to BH3-mimetic drugs and reconciled the disparate pattern of initial clinical response to venetoclax, followed by subsequent treatment failure among patients with TP53-mutant chronic lymphocytic leukemia or acute myeloid leukemia. In contrast to BH3-mimetics targeting just BCL-2 or MCL-1 at doses that are in idually sublethal, a combined BH3-mimetic approach targeting both prosurvival proteins enhanced lethality and durably suppressed the leukemia burden, regardless of TP53 mutation status. Our findings highlight the importance of using sufficiently lethal treatment strategies to maximize outcomes of patients with TP53-mutant disease. In addition, our findings caution against use of sublethal BH3-mimetic drug regimens that may enhance the risk of disease progression driven by emergent TP53-mutant clones.
Publisher: American Society of Hematology
Date: 18-01-2022
DOI: 10.1182/BLOODADVANCES.2021006211
Abstract: The genomic landscape of resistance to targeted agents (TAs) used as monotherapy in chronic lymphocytic leukemia (CLL) is complex and often heterogeneous at the patient level. To gain insight into the clonal architecture of acquired genomic resistance to Bruton tyrosine kinase (BTK) inhibitors and B-cell lymphoma 2 (BCL2) inhibitors in CLL, particularly in patients carrying multiple resistance mutations, we performed targeted single-cell DNA sequencing of 8 patients who developed progressive disease (PD) on TAs (either class). In all cases, analysis of single-cell architecture revealed mutual exclusivity between multiple resistance mutations to the same TA class, variable clonal co-occurrence of multiple mutations affecting different TAs in patients exposed to both classes, and a phenomenon of multiple independent emergences of identical nucleotide changes leading to canonical resistance mutations. We also report the first observation of established BCL2 resistance mutations in a patient with mantle cell lymphoma (MCL) following PD on sequential monotherapy, implicating BCL2 as a venetoclax resistance mechanism in MCL. Taken together, these data reveal the significant clonal complexity of CLL and MCL progression on TAs at the nucleotide level and confirm the presence of multiple, clonally independent, mechanisms of TA resistance within each in idual disease context.
Publisher: Springer Science and Business Media LLC
Date: 29-08-2013
Publisher: Wiley
Date: 13-02-2023
Abstract: Preventing or overcoming resistance to the Bcl‐2 inhibitor venetoclax is an emerging unmet clinical need in patients with chronic lymphocytic leukaemia (CLL). The upregulation of anti‐apoptotic Bcl‐2 members through signalling pathways within the tumor microenvironment appears as a major factor leading to resistance to venetoclax. Previously, we reported that T cells can drive resistance through CD40 and non‐canonical NF‐κB activation and subsequent Bcl‐XL induction. Moreover, the T cell‐derived cytokines IL‐21 and IL‐4 differentially affect Bcl‐XL expression and sensitivity to venetoclax via unknown mechanisms. Here, we mechanistically dissected how Bcl‐XL is regulated in the context of JAK–STAT signalling in primary CLL. First, we demonstrated a clear antagonistic role of IL‐21/STAT3 signalling in the NF‐κB‐mediated expression of Bcl‐XL, whereas IL‐4/STAT6 further promoted the expression of Bcl‐XL. In comparison, Bfl‐1, another NF‐κB target, was not differentially affected by either cytokine. Second, STAT3 and STAT6 affected Bcl‐XL transcription by binding to its promoter without disrupting the DNA‐binding activity of NF‐κB. Third, in situ proximity ligation assays (isPLAs) indicated crosstalk between JAK–STAT signalling and NF‐κB, in which STAT3 inhibited canonical NF‐κB by accelerating nuclear export, and STAT6 promoted non‐canonical NF‐κB. Finally, NF‐κB inducing kinase (NIK) inhibition interrupted the NF‐κB/STAT crosstalk and resensitized CLL cells to venetoclax. In conclusion, we uncovered distinct crosstalk mechanisms that shape the NF‐κB response in CLL towards venetoclax sensitivity or resistance via Bcl‐XL, thereby revealing new potential therapeutic targets.
Publisher: American Society of Hematology
Date: 10-02-2022
Publisher: MDPI AG
Date: 12-11-2020
Abstract: The discovery of the link between defective apoptotic regulation and cancer cell survival engendered the idea of targeting aberrant components of the apoptotic machinery for cancer therapy. The intrinsic pathway of apoptosis is tightly controlled by interactions amongst members of three distinct subgroups of the B-cell lymphoma 2 (BCL2) family of proteins. The pro-survival BCL2 proteins prevent apoptosis by keeping the pro-apoptotic effector proteins BCL2-associated X protein (BAX) and BCL2 homologous antagonist/killer (BAK) in check, while the BH3-only proteins initiate apoptosis by either neutralizing the pro-survival BCL2 proteins or directly activating the pro-apoptotic effector proteins. This tripartite regulatory mechanism is commonly perturbed in B-cell malignancies facilitating cell death evasion. Over the past two decades, structure-based drug discovery has resulted in the development of a series of small molecules that mimic the function of BH3-only proteins called the BH3 mimetics. The most clinically advanced of these is venetoclax, which is a highly selective inhibitor of BCL2 that has transformed the treatment landscape for chronic lymphocytic leukemia (CLL). Other BH3 mimetics, which selectively target myeloid cell leukemia 1 (MCL1) and B-cell lymphoma extra large (BCLxL), are currently under investigation for use in erse malignancies. Here, we review the current role of BH3 mimetics in the treatment of CLL and other B-cell malignancies and address open questions in this rapidly evolving field.
Location: Australia
Start Date: 2017
End Date: 2020
Funder: Leukemia and Lymphoma Society
View Funded ActivityStart Date: 2021
End Date: 2020
Funder: National Health and Medical Research Council
View Funded ActivityStart Date: 2022
End Date: 2024
Funder: Victorian Cancer Agency
View Funded ActivityStart Date: 2024
End Date: 2028
Funder: European Commission
View Funded ActivityStart Date: 2022
End Date: 2024
Funder: Leukaemia Foundation
View Funded Activity