Search

Oncogene-induced DNA damage elicits genomic instability in epithelial cancer cells, but apoptosis is blocked through inactivation of the tumor suppressor p53. In hematological cancers, the relevance of ongoing DNA damage and the mechanisms by which apoptosis is suppressed are largely unknown. We found pervasive DNA damage in hematologic malignancies, including multiple myeloma, lymphoma and leukemia, which leads to activation of a p53-independent, proapoptotic network centered on nuclear relocalization of ABL1 kinase. Although nuclear ABL1 triggers cell death through its interaction with the Hippo pathway coactivator YAP1 in normal cells, we show that low YAP1 levels prevent nuclear ABL1-induced apoptosis in these hematologic malignancies. YAP1 is under the control of a serine-threonine kinase, STK4. Notably, genetic inactivation of STK4 restores YAP1 levels, triggering cell death in vitro and in vivo. Our data therefore identify a new synthetic-lethal strategy to selectively target cancer cells presenting with endogenous DNA damage and low YAP1 levels., In epithelial cancers, rampant DNA double-strand break (DSB) formation followed by activation of the DNA damage response (DDR) occurs in both premalignant and malignant conditions. However, in precancerous settings, senescence [...]

Chronic Lymphocytic Leukemia (CLL) is a malignancy of mature B lymphocytes which are highly dependent on interactions with the tissue microenvironment for their survival and proliferation. Critical components of the microenvironment are monocyte-derived nurselike cells (NLCs), mesenchymal stromal cells, T cells and NK cells, which communicate with CLL cells through a complex network of adhesion molecules, chemokine receptors, tumor necrosis factor (TNF) family members, and soluble factors. (Auto-) antigens and/or autonomous mechanisms activate the B-cell receptor (BCR) and its downstream signaling cascade in secondary lymphatic tissues, playing a central pathogenetic role in CLL. Novel small molecule inhibitors, including the Bruton's tyrosine kinase (BTK) inhibitor ibrutinib and the phosphoinositide-3-kinase delta (PI3Kδ) inhibitor idelalisib, target BCR signaling and have become the most successful new therapeutics in this disease. We here review the cellular and molecular characteristics of CLL cells, and discuss the cellular components and key pathways involved in the cross-talk with their microenvironment. We also highlight the relevant novel treatment strategies, focusing on immunomodulatory agents and BCR signaling inhibitors and how these treatments disrupt CLL–microenvironment interactions. This article is part of a Special Issue entitled: Tumor Microenvironment Regulation of Cancer Cell Survival, Metastasis, Inflammation, and Immune Surveillance edited by Peter Ruvolo and Gregg L. Semenza. [ABSTRACT FROM AUTHOR]

Chronic lymphocytic leukemia (CLL) is characterized by the accumulation in primary and secondary lymphoid tissues of CD5+ B cells that have the same B cell receptor (BCR) rearrangement. Genetic alterations and different stimuli originating from the microenvironment cooperate in the selection and expansion of the malignant clone. Molecular and functional analyses suggest that stimulation through the BCR affects the destiny of leukemic cells in terms of life or death. Microenvironmental signals are crucial for this process, inducing proliferation and leading to the survival and accumulation of leukemic cells within lymphoid organs. Nevertheless, a number of major biological issues still remain to be solved, including the relationships between cell proliferation and cell accumulation within lymphoid organs as well as the mechanisms that regulate CLL cell migration and recirculation between peripheral blood and lymphoid tissues. We focused on the role played by the cytoskeleton, given its relevance in controlling cellular shape, mobility, and homing. We hypothesize that hematopoietic cell-specific Lyn substrate 1 (HS1), a putative prognostic marker in CLL that interacts with distinct cytoskeleton adapters in leukemic B-lymphocytes, could regulate the CLL cell cytoskeleton. [ABSTRACT FROM AUTHOR]

Despite the recent advances in the therapeutic management of Chronic Lymphocytic Leukemia (CLL) patients, this common B cell malignancy still remains incurable. This SnapShot provides an overview of CLL biology and therapy, with a focus on genetics and microenvironmental interactions, which contribute to disease progression and therapy resistance. To view this SnapShot, open or download the PDF. [ABSTRACT FROM AUTHOR]

Mitochondrial apoptosis can be effectively targeted in lymphoid malignancies with the FDA-approved B cell lymphoma 2 (BCL-2) inhibitor venetoclax, but resistance to this agent is emerging. We show that venetoclax resistance in chronic lymphocytic leukemia is associated with complex clonal shifts. To identify determinants of resistance, we conducted parallel genome-scale screens of the BCL-2-driven OCI-Ly1 lymphoma cell line after venetoclax exposure along with integrated expression profiling and functional characterization of drug-resistant and engineered cell lines. We identified regulators of lymphoid transcription and cellular energy metabolism as drivers of venetoclax resistance in addition to the known involvement by BCL-2 family members, which were confirmed in patient samples. Our data support the implementation of combinatorial therapy with metabolic modulators to address venetoclax resistance. • B-lymphoid cancer cells can escape to venetoclax by overexpressing MCL-1 • Modulation of AMPK/PKA axis and lymphoid transcription drive venetoclax resistance • Venetoclax resistance involves changes in cellular energy metabolism such as OXPHOS • Metabolic modulators can cooperate with venetoclax to overcome resistance Guièze et al. show that resistance to the BCL-2 inhibitor venetoclax in chronic lymphocytic leukemia is associated with complex clonal shifts and identify, in addition to the known involvement by BCL-2 family members, regulators of lymphoid transcription and cellular energy metabolism as resistance drivers. [ABSTRACT FROM AUTHOR]

Competing Interests: Declaration of interest ETH has no conflicts of interest to declare. BE has received scientific grants form the companies Abbvie, AstraZeneca, BeiGene, Janssen and Roche. She received honoraria or was participating at advisory boards for Abbvie, AstraZeneca, BeiGene, BMS, Janssen, Lilly, MSD and Roche.

Murine models are indispensable tools for functional genomic studies and preclinical testing of novel therapeutic approaches. Mitochondrial single-cell assay for transposase-accessible chromatin using sequencing (mtscATAC-seq) enables the dissection of cellular heterogeneity and clonal dynamics by capturing chromatin accessibility, copy-number variations (CNV), and mitochondrial DNA (mtDNA) mutations, yet its applicability to murine studies remains unexplored. By leveraging mtscATAC-seq in novel chronic lymphocytic leukemia and Richter syndrome mouse models, we report the detection of mtDNA mutations, particularly in highly proliferative murine cells, alongside CNV and chromatin state changes indicative of clonal evolution upon secondary transplant. This study thus demonstrates the feasibility and utility of multi-modal single-cell and natural barcoding approaches to characterize murine cancer models., Significance: mtDNA mutations can serve as natural barcodes to enable lineage tracing in murine cancer models, which can be used to provide new insights into disease biology and to identify therapeutic vulnerabilities., (©2022 American Association for Cancer Research.)

Transformation to aggressive disease histologies generates formidable clinical challenges across cancers, but biological insights remain few. We modeled the genetic heterogeneity of chronic lymphocytic leukemia (CLL) through multiplexed in vivo CRISPR-Cas9 B-cell editing of recurrent CLL loss-of-function drivers in mice and recapitulated the process of transformation from indolent CLL into large cell lymphoma [i.e., Richter syndrome (RS)]. Evolutionary trajectories of 64 mice carrying diverse combinatorial gene assortments revealed coselection of mutations in Trp53, Mga, and Chd2 and the dual impact of clonal Mga/Chd2 mutations on E2F/MYC and interferon signaling dysregulation. Comparative human and murine RS analyses demonstrated tonic PI3K signaling as a key feature of transformed disease, with constitutive activation of the AKT and S6 kinases, downmodulation of the PTEN phosphatase, and convergent activation of MYC/PI3K transcriptional programs underlying enhanced sensitivity to MYC/mTOR/PI3K inhibition. This robust experimental system presents a unique framework to study lymphoid biology and therapy., Significance: Mouse models reflective of the genetic complexity and heterogeneity of human tumors remain few, including those able to recapitulate transformation to aggressive disease histologies. Herein, we model CLL transformation into RS through multiplexed in vivo gene editing, providing key insight into the pathophysiology and therapeutic vulnerabilities of transformed disease. This article is highlighted in the In This Issue feature, p. 101., (© 2022 American Association for Cancer Research.)

Rapid advances in large-scale next-generation sequencing studies of human samples have progressively defined the highly heterogeneous genetic landscape of chronic lymphocytic leukemia (CLL). At the same time, the numerous challenges posed by the difficulties in rapid manipulation of primary B cells and the paucity of CLL cell lines have limited the ability to interrogate the function of the discovered putative disease "drivers," defined in human sequencing studies through statistical inference. Mouse models represent a powerful tool to study mechanisms of normal and malignant B-cell biology and for preclinical testing of novel therapeutics. Advances in genetic engineering technologies, including the introduction of conditional knockin/knockout strategies, have opened new opportunities to model genetic lesions in a B-cell-restricted context. These new studies build on the experience of generating the MDR mice, the first example of a genetically faithful CLL model, which recapitulates the most common genomic aberration of human CLL: del(13q). In this review, we describe the application of mouse models to the studies of CLL pathogenesis and disease transformation from an indolent to a high-grade malignancy (ie, Richter syndrome [RS]) and treatment, with a focus on newly developed genetically inspired mouse lines modeling recurrent CLL genetic events. We discuss how these novel mouse models, analyzed using new genomic technologies, allow the dissection of mechanisms of disease evolution and response to therapy with greater depth than previously possible and provide important insight into human CLL and RS pathogenesis and therapeutic vulnerabilities. These models thereby provide valuable platforms for functional genomic analyses and treatment studies., (© 2021 by The American Society of Hematology.)

Chronic lymphocytic leukemia (CLL) is characterized by disordered DNA methylation, suggesting these epigenetic changes might play a critical role in disease onset and progression. The methyltransferase DNMT3A is a key regulator of DNA methylation. Although DNMT3A somatic mutations in CLL are rare, we found that low DNMT3A expression is associated with more aggressive disease. A conditional knockout mouse model showed that homozygous depletion of Dnmt3a from B cells results in the development of CLL with 100% penetrance at a median age of onset of 5.3 months, and heterozygous Dnmt3a depletion yields a disease penetrance of 89% with a median onset at 18.5 months, confirming its role as a haploinsufficient tumor suppressor. B1a cells were confirmed as the cell of origin of disease in this model, and Dnmt3a depletion resulted in focal hypomethylation and activation of Notch and Myc signaling. Amplification of chromosome 15 containing the Myc gene was detected in all CLL mice tested, and infiltration of high- Myc -expressing CLL cells in the spleen was observed. Notably, hyperactivation of Notch and Myc signaling was exclusively observed in the Dnmt3a CLL mice, but not in three other CLL mouse models tested ( Sf3b1-Atm , Ikzf3 , and MDR ), and Dnmt3a -depleted CLL were sensitive to pharmacologic inhibition of Notch signaling in vitro and in vivo . Consistent with these findings, human CLL samples with lower DNMT3A expression were more sensitive to Notch inhibition than those with higher DNMT3A expression. Altogether, these results suggest that Dnmt3a depletion induces CLL that is highly dependent on activation of Notch and Myc signaling. SIGNIFICANCE: Loss of DNMT3A expression is a driving event in CLL and is associated with aggressive disease, activation of Notch and Myc signaling, and enhanced sensitivity to Notch inhibition., (©2021 American Association for Cancer Research.)

Hotspot mutation of IKZF3 (IKZF3-L162R) has been identified as a putative driver of chronic lymphocytic leukemia (CLL), but its function remains unknown. Here, we demonstrate its driving role in CLL through a B cell-restricted conditional knockin mouse model. Mutant Ikzf3 alters DNA binding specificity and target selection, leading to hyperactivation of B cell receptor (BCR) signaling, overexpression of nuclear factor κB (NF-κB) target genes, and development of CLL-like disease in elderly mice with a penetrance of ~40%. Human CLL carrying either IKZF3 mutation or high IKZF3 expression was associated with overexpression of BCR/NF-κB pathway members and reduced sensitivity to BCR signaling inhibition by ibrutinib. Our results thus highlight IKZF3 oncogenic function in CLL via transcriptional dysregulation and demonstrate that this pro-survival function can be achieved by either somatic mutation or overexpression of this CLL driver. This emphasizes the need for combinatorial approaches to overcome IKZF3-mediated BCR inhibitor resistance., Competing Interests: Declaration of interests C.J.W. is an equity holder of Biontech, Inc. and receives research funding from Pharmacyclics. D.S.N. has been a consultant for H3 Biomedicine and received research funding from Celgene. J.A.B. reports receiving grant support and advisory board fees from Pharmacyclics, grant support, advisory board fees, and lecture fees from Gilead, advisory board fees from AstraZeneca, and lecture fees and travel support from Janssen. T.J.K. has received research funding and/or has served as an advisor to Ascerta/AstraZeneca, Celgene, Genentech/Roche, Gilead, Janssen, Loxo Oncology, Octernal Therapeutics, Pharmacyclics/AbbVie, TG Therapeutics, VelosBio, and Verastem. Cirmtuzumab was developed by T.J.K. and licensed by the University of California to Oncternal Therapeutics, Inc., which has provided stock/options to the university and T.J.K. All other authors do not have any relevant conflict of interest., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The tumor microenvironment in leukemia and solid tumors induces a shift of activated CD8 + cytotoxic T cells to an exhausted state, characterized by loss of proliferative capacity and impaired immunologic synapse formation. Efficient strategies and targets need to be identified to overcome T-cell exhaustion and further improve overall responses in the clinic. Here, we took advantage of the Eμ-TCL1 chronic lymphocytic leukemia (CLL) and B16 melanoma mouse models to assess the role of the homophilic cell-surface receptor SLAMF6 as an immune-checkpoint regulator. The transfer of SLAMF6 + Eμ-TCL1 cells into SLAMF6 -/- recipients, in contrast to wild-type (WT) recipients, significantly induced expansion of a PD-1 + subpopulation among CD3 + CD44 + CD8 + T cells, which had impaired cytotoxic functions. Conversely, administering anti-SLAMF6 significantly reduced the leukemic burden in Eμ-TCL1 recipient WT mice concomitantly with a loss of PD-1 + CD3 + CD44 + CD8 + T cells with significantly increased effector functions. Anti-SLAMF6 significantly reduced leukemic burden in the peritoneal cavity, a niche where antibody-dependent cellular cytotoxicity (ADCC) is impaired, possibly through activation of CD8 + T cells. Targeting of SLAMF6 affected tumor growth not only in B cell-related leukemia and lymphomas but also in nonhematopoietic tumors such as B16 melanoma, where SLAMF6 is not expressed. In vitro exhausted CD8 + T cells showed increased degranulation when anti-human SLAMF6 was added in culture. Taken together, anti-SLAMF6 both effectively corrected CD8 + T-cell dysfunction and had a direct effect on tumor progression. The outcomes of our studies suggest that targeting SLAMF6 is a potential therapeutic strategy., (©2019 American Association for Cancer Research.)

SF3B1 is recurrently mutated in chronic lymphocytic leukemia (CLL), but its role in the pathogenesis of CLL remains elusive. Here, we show that conditional expression of Sf3b1-K700E mutation in mouse B cells disrupts pre-mRNA splicing, alters cell development, and induces a state of cellular senescence. Combination with Atm deletion leads to the overcoming of cellular senescence and the development of CLL-like disease in elderly mice. These CLL-like cells show genome instability and dysregulation of multiple CLL-associated cellular processes, including deregulated B cell receptor signaling, which we also identified in human CLL cases. Notably, human CLLs harboring SF3B1 mutations exhibit altered response to BTK inhibition. Our murine model of CLL thus provides insights into human CLL disease mechanisms and treatment., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The identification of targetable vulnerabilities in the context of therapeutic resistance is a key challenge in cancer treatment. We detected pervasive aberrant splicing as a characteristic feature of chronic lymphocytic leukemia (CLL), irrespective of splicing factor mutation status, which was associated with sensitivity to the spliceosome modulator, E7107. Splicing modulation affected CLL survival pathways, including members of the B cell lymphoma-2 (BCL2) family of proteins, remodeling antiapoptotic dependencies of human and murine CLL cells. E7107 treatment decreased myeloid cell leukemia-1 (MCL1) dependence and increased BCL2 dependence, sensitizing primary human CLL cells and venetoclax-resistant CLL-like cells from an Eμ-TCL1-based adoptive transfer murine model to treatment with the BCL2 inhibitor venetoclax. Our data provide preclinical rationale to support the combination of venetoclax with splicing modulators to reprogram apoptotic dependencies in CLL for treating venetoclax-resistant CLL cases.

The Bruton's tyrosine kinase (BTK) inhibitor ibrutinib, which antagonizes B cell receptor (BCR) signals, demonstrates remarkable clinical activity in chronic lymphocytic leukemia (CLL). The lymphocytosis experienced by most patients under ibrutinib has previously been attributed to inhibition of BTK-dependent integrin and chemokine cues operating to retain the tumor cells in nodal compartments. Here, we show that the VLA-4 integrin, as expressed by CD49d-positive CLL, can be inside-out activated upon BCR triggering, thus reinforcing the adhesive capacities of CLL cells. In vitro and in vivo ibrutinib treatment, although reducing the constitutive VLA-4 activation and cell adhesion, can be overcome by exogenous BCR triggering in a BTK-independent manner involving PI3K. Clinically, in three independent ibrutinib-treated CLL cohorts, CD49d expression identifies cases with reduced lymphocytosis and inferior nodal response and behaves as independent predictor of shorter progression-free survival, suggesting the retention of CD49d-expressing CLL cells in tissue sites via activated VLA-4. Evaluation of CD49d expression should be incorporated in the characterization of CLL undergoing therapy with BCR inhibitors., (© 2018 Tissino et al.)

In lymphoid organs, nurse-like cells (NLCs) show properties of tumor-associated macrophages, playing a crucial role in chronic lymphocytic leukemia (CLL) cell survival. Ibrutinib, a potent inhibitor of Bruton's tyrosine kinase (BTK), is able to counteract pro-survival signals in CLL cells. Since the effects on CLL cells have been studied in the last years, less is known about the influence of ibrutinib on NLCs properties. We sought to determine how ibrutinib modifies NLCs functions focusing on the balance between immunosuppressive and inflammatory features. Our data show that ibrutinib targets BTK expressed by NLCs modifying their phenotype and function. Treatment with ibrutinib reduces the phagocytic ability and increases the immunosuppressive profile of NLCs exacerbating the expression of M2 markers. Accordingly, ibrutinib hampers LPS-mediated signaling, decreasing STAT1 phosphorylation, while allows IL-4-mediated STAT6 phosphorylation. In addition, NLCs treated with ibrutinib are able to protect CLL cells from drug-induced apoptosis partially through the secretion of IL-10. Results from patient samples obtained prior and after 1 month of treatment with ibrutinib show an accentuation of CD206, CD11b and Tie2 in the monocytic population in the peripheral blood. Our study provides new insights into the immunomodulatory action of ibrutinib on monocyte/macrophage population in CLL.

BCR signaling is a central pathogenetic pathway in chronic lymphocytic leukemia (CLL). Most CLL cells express BCRs of IgM and IgD isotypes, but the contribution of these isotypes to functional responses remains incompletely defined. We therefore investigated differences between IgM and IgD signaling in freshly isolated peripheral blood CLL cells and in CLL cells cultured with nurselike cells, a model that mimics the lymph node microenvironment. IgM signaling induced prolonged activation of ERK kinases and promoted CLL cell survival, CCL3 and CCL4 chemokine secretion, and downregulation of BCL6, the transcriptional repressor of CCL3 In contrast, IgD signaling induced activation of the cytoskeletal protein HS1, along with F-actin polymerization, which resulted in rapid receptor internalization and failure to support downstream responses, including CLL cell survival and chemokine secretion. IgM and IgD receptor downmodulation, HS1 and ERK activation, chemokine secretion, and BCL6 downregulation were also observed when CLL cells were cocultured with nurselike cells. The Bruton's tyrosine kinase inhibitor ibrutinib effectively inhibited both IgM and IgD isotype signaling. In conclusion, through a variety of functional readouts, we demonstrate very distinct outcomes of IgM and IgD isotype activation in CLL cells, providing novel insight into the regulation of BCR signaling in CLL., (Copyright © 2016 by The American Association of Immunologists, Inc.)

Purpose: We evaluated efficacy and tolerability of the combination of ofatumumab and lenalidomide in patients with relapsed/refractory chronic lymphocytic leukemia (CLL), and explored whether immune system characteristics could influence the response to treatment., Experimental Design: Thirty-four patients were enrolled in this phase II study. Ofatumumab was administered at a dose of 300 mg on day 1, 1,000 mg on days 8, 15, and 22 during course 1, 1,000 mg on day 1 during courses 3-6, and once every other course during courses 7-24 (28-day courses). Oral lenalidomide (10 mg daily) was started on day 9 and continued for as long as a clinical benefit was observed., Results: The overall response rate was 71%. Eight patients (24%) achieved a complete remission (CR) or CR with incomplete recovery of blood counts, including 9% with minimal residual disease-negative CR. The median progression-free survival was 16 months, and the estimated 5-year survival was 53%. The most common treatment-related toxicity was neutropenia (grade >2 in 18% of the 574 patient courses). The most frequent infectious complications were pneumonia and neutropenic fever (24% and 9% of patients, respectively). We observed that patients who achieved a CR had at baseline higher numbers and a better preserved function of T cells and natural killer cells compared with non-responders., Conclusions: The combination of ofatumumab and lenalidomide is a well-tolerated regimen that induces durable responses in the majority of patients with relapsed/refractory CLL. Our correlative data suggest a role of competent immune system in supporting the efficacy of this treatment. Clin Cancer Res; 22(10); 2359-67. ©2016 AACR., Competing Interests: of Potential Conflicts of Interest: S. O'Brien is a consultant/advisory board member for Celgene. J.M. Reuben reports receiving a commercial research grant from Hitachi Chemical Co. and is a consultant/advisory board member for Angle, PLC, and Hitachi Chemical Co. No potential conflicts of interest were disclosed by the other authors., (©2016 American Association for Cancer Research.)