Search

•T-bet acts as a tumor suppressor by enhancing interferon signaling and suppressing proliferation of malignant B cells.•T-bet expression in CLL cells is positively correlated with longer overall survival in patients with CLL.

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.

The deletion of 11q (del(11q)) invariably comprises ATMgene in chronic lymphocytic leukemia (CLL). Concomitant mutations in this gene in the remaining allele have been identified in 1/3 of CLL cases harboring del(11q), being the biallelic loss of ATMassociated with adverse prognosis. Although the introduction of targeted BCR inhibition has significantly favored the outcomes of del(11q) patients, responses of patients harboring ATM functional loss through biallelic inactivation are unexplored, and the development of resistances to targeted therapies have been increasingly reported, urging the need to explore novel therapeutic approaches. Here, we generated isogenic CLL cell lines harboring del(11q) and ATMmutations through CRISPR/Cas9-based gene-editing. With these models, we uncovered a novel therapeutic vulnerability of del(11q)/ATM-mutated cells to dual BCR and PARP inhibition. Ex vivo studies in the presence of stromal stimulation on 38 CLL primary samples confirmed a synergistic action of the combination of olaparib and ibrutinib in del(11q)/ATM-mutated CLL patients. In addition, we showed that ibrutinib produced a homologous recombination repair impairment through RAD51 dysregulation, finding a synergistic link of both drugs in the DNA damage repair pathway. Our data provide a preclinical rationale for the use of this combination in CLL patients with this high-risk cytogenetic abnormality.

Despite major improvements in treatment outcome with novel targeted therapies, such as the Bruton tyrosine kinase (BTK) inhibitor ibrutinib, chronic lymphocytic leukemia (CLL) remains incurable in the majority of patients. Activation of PI3K, NF-κB, and/or MYC has been linked to residual disease and/or resistance in ibrutinib-treated patients. These pathways can be targeted by inhibitors of bromodomain and extra-terminal (BET) proteins. Here we report about the preclinical activity of GS-5829, a novel BET inhibitor, in CLL. GS-5829 inhibited CLL cell proliferation and induced leukemia cell apoptosis through deregulation of key signaling pathways, such as BLK, AKT, ERK1/2, and MYC. IκBα modulation indicates that GS-5829 also inhibited NF-κB signaling. GS-5829-induced apoptosis resulted from an imbalance between positive (BIM) and negative regulators (BCL-XL) of the intrinsic apoptosis pathway. The antileukemia activity of GS-5829 increased synergistically in combinations with B-cell receptor signaling inhibitors, the BTK inhibitor ibrutinib, the PI3Kδ inhibitor idelalisib, and the SYK inhibitor entospletinib. In cocultures that mimic the lymph node microenvironment, GS-5829 inhibited signaling pathways within nurselike cells and their growth, indicating that BET inhibitors also can target the supportive CLL microenvironment. Collectively, these data provide a rationale for the clinical evaluation of BET inhibitors in CLL.

B cell receptor (BCR) signaling is a central pathway promoting the survival and proliferation of normal and malignant B cells. Chronic lymphocytic leukemia (CLL) arises from mature B cells, expressing functional BCRs, mainly of immunoglobulin M (IgM) and IgD isotypes. Importantly, 30% of CLL patients express quasi-identical BCRs, the so-called “stereotyped” receptors, indicating the existence of common antigenic determinants, which may drive disease initiation and favor its progression. Although the antigenic specificity of IgM and IgD receptors is identical, there are distinct isotype-specific responses after IgM and IgD triggering. Here, we discuss the most important steps of normal B cell development, and highlight the importance of BCR signaling for CLL pathogenesis, with a focus on differences between IgM and IgD isotype signaling. We also highlight the main characteristics of CLL patient subsets, based on BCR stereotypy, and describe subset-specific BCR function and antigen-binding characteristics. Finally, we outline the key biologic and clinical responses to kinase inhibitor therapy, targeting the BCR-associated Bruton’s tyrosine kinase, phosphoinositide-3-kinase, and spleen tyrosine kinase in patients with CLL.

Here, we present a protocol to generate B cell restricted mouse models of loss-of-function genetic drivers typical of lymphoproliferative disorders, using stem cell engineering of murine strains carrying B cell restricted Cas9 expression. We describe steps for preparing lentivirus expressing sgRNA-mCherry, isolating hematopoietic stem and progenitor cells, and in vitrotransduction. We then detail the transplantation of engineered cells into recipient mice and verification of gene edits. These mouse models represent versatile platforms to model complex disease traits typical of lymphoproliferative disorders.

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.

While the genetic landscape of chronic lymphocytic leukemia (CLL) has been broadly profiled by large-scale sequencing studies performed over the past decade, the molecular basis of the transformation of CLL to aggressive lymphoma, or Richter syndrome (RS), has remained incompletely characterized. Recent advances in computational methods of clonal deconvolution, as well as extensive sample collection efforts in this rapidly progressive malignancy, have now enabled comprehensive analysis of paired CLL and RS samples and have led to multiple new studies investigating the genetic, transcriptomic and epigenetic origins of RS. In parallel, new genetically-engineered and xenograft mouse models have provided the opportunity for gleaning fresh biologic and mechanistic insights into RS development and stepwise evolution from antecedent CLL. Altogether, these studies have defined RS driver lesions, CLL risk lesions and identified pathways dysregulated in transformation. Moreover, unique molecular subtypes of RS have been revealed, including disease marked by profound genomic instability with chromothripsis/chromoplexy and whole genome duplication. Novel profiling approaches, including single-cell DNA and transcriptome sequencing of RS biopsy specimens and cell-free DNA profiling of patient plasma, demonstrate promise for the timely identification of RS clones and may translate to non-invasive identification and early diagnosis of RS. This review summarizes the recent scientific advances in RS and supports the integrated study of human genomics with mouse modeling to provide advanced understanding of the biologic underpinnings of transformation. These recent studies have major implications for much needed novel therapeutic strategies for this still largely incurable malignancy.

Hypoxia-inducible transcription factors (HIFs) regulate a wide array of adaptive responses to hypoxia and are often activated in solid tumors and hematologic malignancies due to intratumoral hypoxia and emerging new layers of regulation. We found that in chronic lymphocytic leukemia (CLL), HIF-1α is a novel regulator of the interaction of CLL cells with protective leukemia microenvironments and, in turn, is regulated by this interaction in a positive feedback loop that promotes leukemia survival and propagation. Through unbiased microarray analysis, we found that in CLL cells, HIF-1α regulates the expression of important chemokine receptors and cell adhesion molecules that control the interaction of leukemic cells with bone marrow and spleen microenvironments. Inactivation of HIF-1α impairs chemotaxis and cell adhesion to stroma, reduces bone marrow and spleen colonization in xenograft and allograft CLL mouse models, and prolongs survival in mice. Of interest, we found that in CLL cells, HIF-1α is transcriptionally regulated after coculture with stromal cells. Furthermore, HIF-1α messenger RNA levels vary significantly within CLL patients and correlate with the expression of HIF-1α target genes, including CXCR4, thus further emphasizing the relevance of HIF-1α expression to CLL pathogenesis.

Hypoxia-inducible transcription factors (HIFs) regulate a wide array of adaptive responses to hypoxia and are often activated in solid tumors and hematologic malignancies due to intratumoral hypoxia and emerging new layers of regulation. We found that in chronic lymphocytic leukemia (CLL), HIF-1α is a novel regulator of the interaction of CLL cells with protective leukemia microenvironments and, in turn, is regulated by this interaction in a positive feedback loop that promotes leukemia survival and propagation. Through unbiased microarray analysis, we found that in CLL cells, HIF-1α regulates the expression of important chemokine receptors and cell adhesion molecules that control the interaction of leukemic cells with bone marrow and spleen microenvironments. Inactivation of HIF-1α impairs chemotaxis and cell adhesion to stroma, reduces bone marrow and spleen colonization in xenograft and allograft CLL mouse models, and prolongs survival in mice. Of interest, we found that in CLL cells, HIF-1α is transcriptionally regulated after coculture with stromal cells. Furthermore, HIF-1α messenger RNA levels vary significantly within CLL patients and correlate with the expression of HIF-1α target genes, including CXCR4, thus further emphasizing the relevance of HIF-1α expression to CLL pathogenesis.

B-cell receptor (BCR) triggering and responsiveness have a crucial role in the survival and expansion of chronic lymphocytic leukemia (CLL) clones. Analysis of in vitro response of CLL cells to BCR triggering allowed the definition of 2 main subsets of patients and lack of signaling capacity was associated with constitutive activation of extracellular-regulated kinases 1/2 (ERK1/2) and nuclear factor of activated T cells c1 (NF-ATc1), consistent with the idea that at least one group of CLL patients derives from the abnormal expansion of anergic B cells. In the present work, we further investigated the anergic subset of CLL (defined as the one with constitutive ERK1/2 phosphorylation) and found that it is characterized by low levels of surface immunoglobulin M and impairment of calcium mobilization after BCR engagement in vitro. Chronic BCR triggering promoted CLL cell survival selectively in phosphorylated ERK1/2 samples and the use of mitogen-activated protein kinase and NF-AT signaling inhibitors specifically induced apoptosis in this group of patients. Apoptosis induction was preceded by an initial phase of anergy reversal consisting in the loss of ERK phosphorylation and NF-AT nuclear translocation and by the restoration of BCR responsiveness, reinforcing the idea that the anergic program favors the survival of leukemic lymphocytes.

B-cell receptor (BCR) triggering and responsiveness have a crucial role in the survival and expansion of chronic lymphocytic leukemia (CLL) clones. Analysis of in vitro response of CLL cells to BCR triggering allowed the definition of 2 main subsets of patients and lack of signaling capacity was associated with constitutive activation of extracellular-regulated kinases 1/2 (ERK1/2) and nuclear factor of activated T cells c1 (NF-ATc1), consistent with the idea that at least one group of CLL patients derives from the abnormal expansion of anergic B cells. In the present work, we further investigated the anergic subset of CLL (defined as the one with constitutive ERK1/2 phosphorylation) and found that it is characterized by low levels of surface immunoglobulin M and impairment of calcium mobilization after BCR engagement in vitro. Chronic BCR triggering promoted CLL cell survival selectively in phosphorylated ERK1/2 samples and the use of mitogen-activated protein kinase and NF-AT signaling inhibitors specifically induced apoptosis in this group of patients. Apoptosis induction was preceded by an initial phase of anergy reversal consisting in the loss of ERK phosphorylation and NF-AT nuclear translocation and by the restoration of BCR responsiveness, reinforcing the idea that the anergic program favors the survival of leukemic lymphocytes.

HS1 (hematopoietic cell-specific Lyn substrate-1) is a cytoskeletal interactor in the B-cell receptor (BCR) signaling pathway whose phosphorylation correlates with prognosis in Chronic Lymphocytic Leukemia (CLL). The differentially phosphorylated sites and the kinases that regulate HS1 activity in CLL remain poorly understood. We demonstrate that HS1 activity is differentially regulated by LYN kinase that, in a subset of patients, phosphorylates HS1 on Tyrosine (Y)397, resulting in its activation. This correlates with increased cytoskeletal functionality in terms of migration, adhesion and F-actin polymerization. In these patients, LYN is also activated on Y396 residue and its inhibition with the tyrosine kinase inhibitor Dasatinib abrogates HS1-Y397 phosphorylation. This results in the reduction of HS1 activation along with that of cytoskeletal effector VAV1 and the downstream kinase ERK also in the presence of BCR and CXC chemokine receptor CXCR4 stimulation. Interestingly, targeting the LYN/HS1 axis in vitro leads to the concomitant reduction of cytoskeletal activity, BCR signaling and cell survival in the subset of patients with activated LYN/HS1. In a transplantable mouse model based on the EμTCL1 transgenic mouse, LYN/HS1 signaling inhibition interferes with CLL progression and lymphoid organ infiltration. Thus LYN/HS1 axis marks distinct signaling profiles and cytoskeletal-related features that may represent valuable targets for cytoskeleton-targeted therapeutic intervention in CLL.

HS1 (hematopoietic cell-specific Lyn substrate-1) is a cytoskeletal interactor in the B-cell receptor (BCR) signaling pathway whose phosphorylation correlates with prognosis in Chronic Lymphocytic Leukemia (CLL). The differentially phosphorylated sites and the kinases that regulate HS1 activity in CLL remain poorly understood. We demonstrate that HS1 activity is differentially regulated by LYN kinase that, in a subset of patients, phosphorylates HS1 on Tyrosine (Y)397, resulting in its activation. This correlates with increased cytoskeletal functionality in terms of migration, adhesion and F-actin polymerization. In these patients, LYN is also activated on Y396 residue and its inhibition with the tyrosine kinase inhibitor Dasatinib abrogates HS1-Y397 phosphorylation. This results in the reduction of HS1 activation along with that of cytoskeletal effector VAV1 and the downstream kinase ERK also in the presence of BCR and CXC chemokine receptor CXCR4 stimulation. Interestingly, targeting the LYN/HS1 axis in vitro leads to the concomitant reduction of cytoskeletal activity, BCR signaling and cell survival in the subset of patients with activated LYN/HS1. In a transplantable mouse model based on the EμTCL1 transgenic mouse, LYN/HS1 signaling inhibition interferes with CLL progression and lymphoid organ infiltration. Thus LYN/HS1 axis marks distinct signaling profiles and cytoskeletal-related features that may represent valuable targets for cytoskeleton-targeted therapeutic intervention in CLL.

The function of the intracellular protein hematopoietic cell–specific Lyn substrate-1 (HS1) in B lymphocytes is poorly defined. To investigate its role in migration, trafficking, and homing of leukemic B lymphocytes we have used B cells from HS1−/− mice, the HS1-silenced human chronic lymphocytic leukemia (CLL) MEC1 cell line and primary leukemic B cells from patients with CLL. We have used both in vitro and in vivo models and found that the lack of expression of HS1 causes several important functional effects. In vitro, we observed an impaired cytoskeletal remodeling that resulted in diminished cell migration, abnormal cell adhesion, and increased homotypic aggregation. In vivo, immunodeficient Rag2−/−γc−/− mice injected with HS1-silenced CLL B cells showed a decreased organ infiltration with the notable exception of the bone marrow (BM). The leukemic-prone Eμ-TCL1 transgenic mice crossed with HS1-deficient mice were compared with Eμ-TCL1 mice and showed an earlier disease onset and a reduced survival. These findings show that HS1 is a central regulator of cytoskeleton remodeling that controls lymphocyte trafficking and homing and significantly influences the tissue invasion and infiltration in CLL.

The function of the intracellular protein hematopoietic cell–specific Lyn substrate-1 (HS1) in B lymphocytes is poorly defined. To investigate its role in migration, trafficking, and homing of leukemic B lymphocytes we have used B cells from HS1−/−mice, the HS1-silenced human chronic lymphocytic leukemia (CLL) MEC1 cell line and primary leukemic B cells from patients with CLL. We have used both in vitro and in vivo models and found that the lack of expression of HS1 causes several important functional effects. In vitro, we observed an impaired cytoskeletal remodeling that resulted in diminished cell migration, abnormal cell adhesion, and increased homotypic aggregation. In vivo, immunodeficient Rag2−/−γc−/−mice injected with HS1-silenced CLL B cells showed a decreased organ infiltration with the notable exception of the bone marrow (BM). The leukemic-prone Eµ-TCL1 transgenic mice crossed with HS1-deficient mice were compared with Eµ-TCL1 mice and showed an earlier disease onset and a reduced survival. These findings show that HS1 is a central regulator of cytoskeleton remodeling that controls lymphocyte trafficking and homing and significantly influences the tissue invasion and infiltration in CLL.

Neuberg: Pharmacyclics: Research Funding; Celgene: Research Funding; Madrigak Pharmaceuticals: Current equity holder in publicly-traded company. Getz:Broad Institute: Patents & Royalties: MuTect, ABSOLUTE, MutSig, MSMuTect, MSMutSig, POLYSOLVER and TensorQTL; Pharmacyclics: Research Funding; IBM: Research Funding; Scorpion Therapeutics: Consultancy, Current equity holder in publicly-traded company, Other: Founder. Wu:BionTech: Current equity holder in publicly-traded company; Pharmacyclics: Research Funding.

Amongst the novel putative drivers identified by large-scale sequencing studies of chronic lymphocytic leukemia (CLL) is the ribosomal protein RPS15. Mutated in 5.3% of CLL, it co-occurs with heterozygous TP53alterations in 36% of RPS15-mutated samples. Mutation of this mediator of ribosome maturation and translation is associated with poor disease prognosis and enriched in cohorts with del(17p) and relapsed CLL, suggesting a role in disease progression and therapeutic resistance. However, the impact of RPS15mutation on B cell function and CLL development, in the presence or absence of TP53mutation, has yet to be characterized. To this end, we developed overexpression HG3 CLL cell lines modeling four common RPS15mutations (G134R, H137Y, S138F, and S139F) and a conditional knock-in mouse model of the S138F mutation with and without heterozygous Trp53deletion (generated by crossing Rps15and Trp53mutant mice with Cd19-Cre mice).

RNA splicing factor SF3B1is one of the most recurrently mutated genes in chronic lymphocytic leukemia (CLL), but expression of this mutation alone in murine B cells does not result in CLL. This gene mutation is often subclonal and associated with poor survival. How this mutation impacts CLL progression remains elusive. Since SF3B1mutation frequently co-occurs with chromosome 13q deletion (del(13q)), and mice with deletion of the Minimal Deleted Region (MDR) of del(13q) develop indolent CLL, we therefore asked whether co-expression of Sf3b1mutation can accelerate the onset of CLL in this murine model. If so, how does Sf3b1mutation mechanistically contribute to CLL.

Although we have gained a wealth of knowledge from large-scale DNA sequencing studies across blood cancers, we still know little about the functional interplay of the discovered putative drivers in the generation of chronic lymphocytic leukemia (CLL) and its transformation into Richter's syndrome (RS). We have previously observed that CRISPR-Cas9 in vivoB-cell editing of common CLL loss-of-function (LOF) lesions (Atm, Trp53, Chd2, Birc3, Mga, Samhd1) can increase in vitroB cell fitness, but is not sufficient to sustain in vivoB cell survival after 12 months post-transplant.

Richter's syndrome (RS) represents one of the foremost challenges in CLL management, and its pathogenesis remains largely undefined. We recently leveraged CRISPR-Cas9 in vivogene editing to develop mouse models of RS by engineering multiplexed loss-of-function lesions typical of CLL (Atm, Trp53, Chd2, Birc3, Mga, Samhd1) in early stem and progenitor cells [Lineage -Sca-1 +c-kit +(LSK)] from MDR-Cd19Cas9 donor mice. These animals express Cas9-GFP in a B-cell restricted fashion and the leukemogenic MDRlesion, which mimics del(13q) when the sgRNA-transduced LSK cells are transplanted in CD45.1 immunocompetent recipients. Through these methods, we observed not only development of CLL, but also transformation into RS, and even captured a stage where CLL and RS were co-existing in the same animal (CLL/RS).

Previous studies have revealed a critical role of methylation deregulation in the onset and progression of chronic lymphocytic leukemia (CLL). In mammalian cells, DNA methylation is dynamically established by the DNA methyltransferase 3 (DNMT3) family of de novomethyltransferases DNMT3A. Although mutations of DNMT3Aare rarely observed in CLL, our RNA-sequencing (RNA-seq) analysis of 107 human CLLs show that low DNMT3Aexpression is associated with more aggressive disease, and supports a driving role of DNMT3Aloss in CLL. To test this hypothesis, we generated a conditional knock-out mouse model with B cell-restricted deletion of Dnmt3a. Homozygous Dnmt3adepletion in B cells resulted in the development of CD5+ B cell leukemia mimicking human CLL with 100% penetrance at a median age of onset of 5.3 months, and heterozygous Dnmt3adepletion yielded a disease penetrance of 89% with a median onset at 18.5 months, confirming its role as a haplo‐insufficient tumor suppressor.

Davids: Surface Oncology: Research Funding; Eli Lilly and Company: Consultancy; Genentech: Consultancy, Research Funding; Takeda: Consultancy; MEI Pharma: Consultancy; Janssen: Consultancy; Verastem: Consultancy, Research Funding; Ascentage Pharma: Consultancy, Research Funding; Pharmacyclics: Consultancy, Research Funding; TG Therapeutics: Consultancy, Research Funding; Astra-Zeneca: Consultancy, Research Funding; Merck: Consultancy; Adaptive Biotechnologies: Consultancy; Research to Practice: Consultancy; AbbVie: Consultancy; MEI Pharma: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; BMS: Consultancy, Research Funding; Celgene: Consultancy; BeiGene: Consultancy. Letai: Dialectic Therapeutics: Other: equity holding member of the scientific advisory board; Flash Therapeutics: Other: equity holding member of the scientific advisory board; Zentalis Pharmaceuticals: Other: equity holding member of the scientific advisory board. Neuberg: Madrigal Pharmaceuticals: Other: Stock ownership; Pharmacyclics: Research Funding. Wu: Pharmacyclics: Research Funding; BioNTech: Current equity holder in publicly-traded company.

Kipps: Abbott Laboratories: Consultancy, Research Funding; Celgene Corporation: Consultancy, Honoraria, Research Funding; Pharmacyclics LLC, an Abbvie Company: Consultancy, Honoraria, Other: Travel, Accommodations, Expenses, Research Funding, Speakers Bureau; Genentech, Inc.: Honoraria, Research Funding, Speakers Bureau; Gilead Sciences, Inc.: Honoraria, Research Funding; GlaxoSmithKline: Research Funding; MedImmune Inc: Research Funding; Moores Cancer Center: Current Employment; Oncternal Therapeutics, Inc.: Current holder of stock options in a privately-held company, Other: Stock or other ownership, Patents & Royalties: Cirmtuzumab was developed by Thomas J. Kipps in the Thomas J. Kipps laboratory and licensed by the University of California to Oncternal Therapeutics, Inc., which provided stock options and research funding to the Thomas J. Kipps laboratory., Research Funding; AbbVie: Consultancy, Honoraria, Other, Speakers Bureau; DAVAOncology: Consultancy, Honoraria, Other; DAVA Pharmaceuticals: Speakers Bureau; Bionest Partner: Other; Celgene: Consultancy, Honoraria, Other, Research Funding; Genetech: Honoraria, Other; Genentech-Roche: Consultancy; Gilead Sciences: Consultancy, Honoraria, Other, Speakers Bureau; Janssen: Consultancy, Honoraria, Other, Research Funding, Speakers Bureau; Roche: Honoraria, Other; MD Anderson Cancer Center: Research Funding; Velos: Research Funding; CRIM: Research Funding; Indy Hematology Review: Other; TG Therapeutics: Other; Verstem: Other, Speakers Bureau; University of California, San Diego: Current Employment; Pharmacyclics/AbbVie: Honoraria, Research Funding; Breast Cancer Research Foundation: Research Funding; SCOR - The Leukemia and Lymphoma Society: Research Funding; National Cancer Institute/NIH: Honoraria, Research Funding; Genentech/Roche: Honoraria; European Research Initiative on CLL (ERIC): Honoraria. Neuberg: Madrigal Pharmaceuticals: Other: Stock ownership; Pharmacyclics: Research Funding. Letai: Flash Therapeutics: Other: equity holding member of the scientific advisory board; Dialectic Therapeutics: Other: equity holding member of the scientific advisory board; Zentalis Pharmaceuticals: Other: equity holding member of the scientific advisory board. Wu: BioNTech: Current equity holder in publicly-traded company; Pharmacyclics: Research Funding.

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 knock-in/knock-out 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 MDRmice, the first example of a genetically-faithful CLL model and 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 (i.e. Richter's syndrome – RS) and treatment, with a focus on newly developed genetically-inspired mouse lines modelling recurrent CLL genetic events. We discuss how these novel mouse models, analyzedthrough the use of 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.

CRISPR-Cas9 gene editing has transformed our ability to rapidly interrogate the functional impact of somatic mutations in human cancers. Droplet-based technology enables the analysis of Cas9-introduced gene edits in thousands of single cells. Using this technology, we analyze Ba/F3 cells engineered to express single or multiplexed loss-of-function mutations recurrent in chronic lymphocytic leukemia. Our approach reliably quantifies mutational co-occurrences, zygosity status, and the occurrence of Cas9 edits at single-cell resolution.

Kipps: Pharmacyclics/ AbbVie, Breast Cancer Research Foundation, MD Anderson Cancer Center, Oncternal Therapeutics, Inc., Specialized Center of Research (SCOR) - The Leukemia and Lymphoma Society (LLS), California Institute for Regenerative Medicine (CIRM): Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Honoraria, Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Genentech/Roche: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; VelosBio: Research Funding; Oncternal Therapeutics, Inc.: Other: Cirmtuzumab was developed by Thomas J. Kipps in the Thomas J. Kipps laboratory and licensed by the University of California to Oncternal Therapeutics, Inc., which provided stock options and research funding to the Thomas J. Kipps laboratory, Research Funding; Ascerta/AstraZeneca, Celgene, Genentech/F. Hoffmann-La Roche, Gilead, Janssen, Loxo Oncology, Octernal Therapeutics, Pharmacyclics/AbbVie, TG Therapeutics, VelosBio, and Verastem: Membership on an entity's Board of Directors or advisory committees. Wu:BionTech: Current equity holder in publicly-traded company; Pharmacyclics: Research Funding.

A hotspot mutation within the DNA-binding domain of IKZF3(IKZF3-L162R) has been identified as a putative driver in chronic lymphocytic leukemia (CLL); however, its functional effects are unknown. We recently confirmed its role as a CLL driver in a B cell-restricted conditional knock-in model. IKZF3mutation altered mature B cell development and signaling capacity, and induced CLL-like disease in elderly mice (~40% penetrance). Moreover, we found IKZF3-L162R acts as a gain-of-function mutation, altering DNA binding specificity and target selection of IKZF3, and resulting in overexpression of multiple B-cell receptor (BCR) genes. Consistent with the murine data, RNA-sequencing analysis showed that human CLL cells with mut-IKZF3[n=4] have an enhanced signature of BCR-signaling gene expression compared to WT-IKZF3[n=6, all IGHVunmutated] (p<0.001), and also exhibited general upregulation of key BCR-signaling regulators. These results confirm the role of IKZF3as a master regulator of BCR-signaling gene expression, with the mutation contributing to overexpression of these genes.

Guièze: Abbvie: Honoraria; Roche: Honoraria; Janssen: Honoraria; Gilead: Honoraria. Thompson:AbbVie: Research Funding; Amgen: Consultancy, Research Funding; Pfizer: Research Funding; Pharmacyclics: Research Funding; Genentech: Consultancy, Honoraria; Gilead: Consultancy, Honoraria. Davids:AbbVie, Acerta Pharma, Adaptive, Biotechnologies, Astra-Zeneca, Genentech, Gilead Sciences, Janssen, Pharmacyclics, TG therapeutics: Membership on an entity's Board of Directors or advisory committees; Research to Practice: Honoraria; AbbVie, Astra-Zeneca, Genentech, Janssen, MEI, Pharmacyclics, Syros Pharmaceuticals, Verastem: Consultancy; Acerta Pharma, Ascentage Pharma, Genentech, MEI pharma, Pharmacyclics, Surface Oncology, TG Therapeutics, Verastem: Research Funding. Brown:AbbVie: Consultancy; Acerta Pharma: Consultancy; Loxo: Consultancy, Research Funding; BeiGene: Consultancy; Catapult Therapeutics: Consultancy; AstraZeneca: Consultancy; Novartis: Consultancy; Pfizer: Consultancy; Pharmacyclics: Consultancy; Sunesis: Consultancy; TG Therapeutics: Consultancy; Verastem: Consultancy, Research Funding; Sun Pharmaceuticals: Research Funding; Janssen: Honoraria; Teva: Honoraria; Morphosys: Other: Data safety monitoring board; Invectys: Other: Data safety monitoring board; Octapharma: Consultancy; Kite, a Gilead Company: Consultancy, Research Funding; Juno/Celgene: Consultancy; Dynamo Therapeutics: Consultancy; Genentech/Roche: Consultancy; Gilead: Consultancy, Research Funding. Wierda:Xencor: Research Funding; Cyclcel: Research Funding; Genentech: Research Funding; Pharmacyclics LLC: Research Funding; Gilead Sciences: Research Funding; KITE pharma: Research Funding; Oncternal Therapeutics Inc.: Research Funding; Sunesis: Research Funding; AbbVie: Research Funding; Janssen: Research Funding; Acerta Pharma Inc: Research Funding; GSK/Novartis: Research Funding; Miragen: Research Funding; Loxo Oncology Inc.: Research Funding; Juno Therapeutics: Research Funding. Letai:AbbVie, AstraZeneca, Novartis: Consultancy, Research Funding; Zeno Pharmaceuticals, Vivid Bioscience, Flash Therapeutics, Dialectic Therapeutics: Membership on an entity's Board of Directors or advisory committees, Other: Cofounder or Advisory Board member. Neuberg:Pharmacyclics: Research Funding; Madrigal Pharmaceuticals: Equity Ownership; Celgene: Research Funding. Mootha:Jansen Pharmaceuticals: Other: SAB, compensation; 5am Ventures: Other: SAB, compensation; Raze Therapeutics: Other: Founder, SAB, equity. Getz:MuTect, ABSOLTUE, MutSig and POLYSOLVER: Patents & Royalties: MuTect, ABSOLTUE, MutSig and POLYSOLVER; Pharmacyclics: Research Funding; IBM: Research Funding. Wu:Pharmacyclics: Research Funding; Neon Therapeutics: Other: Member, Advisory Board.

Venetoclax, the first approved BH3 mimetic targeting BCL2, demonstrates high response rate in chronic lymphocytic leukemia (CLL) but resistant cases are emerging. Aside from BCL2 mutations affecting venetoclax binding, multiple lines of mounting evidence suggest a role for non-mutational mechanisms underlying resistance to this drug. By applying both CRISPR-Cas9 knock-out and ORF overexpression screens in the lymphoma cell line OCI-Ly1, we previously reported the identification of MCL-1 overexpression and of the AMPK/PKA signaling axis in altering energy metabolism underlying venetoclax resistance (Guieze, ASH 2018). Here, we report further in-depth exploration of the impact of these findings, discovered through the analysis of lymphoid cell lines, and of specimens collected from CLL patients developing venetoclax resistance.

Mouse models that reflect human cancer genetics are valuable tools for dissecting disease mechanisms and for discovering novel therapeutic vulnerabilities, although such models for chronic lymphocytic leukemia (CLL) have been largely lacking until now. The most commonly used CLL model, the TCL1 mouse, is based on constitutive B-cell restricted expression of the human TCL1 oncogene, but does not reflect CLL genetics per se. Novel mouse models, such as the MDR mice (modeling del[13q]) and our recently developed Sf3b1mut/Atmdeldouble mutant mice, are able to recapitulate the expression of CLL driver mutations and develop CLL-like disease with a rather delayed onset (~18 months), mimicking the indolent nature of human CLL.

Mouse models represent invaluable tools for the systematic evaluation of cancer drivers, yet models that address the impact of putative genetic drivers of chronic lymphocytic leukemia (CLL) on B cell development and function are largely lacking.

Valentin: Abbvie Inc: Other: Travel reimbursement; Roche: Other: Travel reimbursement. Kipps:Pharmacyclics: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Membership on an entity's Board of Directors or advisory committees, Research Funding; Genentech, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding; Velos-Bio: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding; Jannsen Pharmaceutical Companies of Johnson & Johnson: Honoraria, Membership on an entity's Board of Directors or advisory committees; AstraZeneca, Inc.: Membership on an entity's Board of Directors or advisory committees; Verastem: Membership on an entity's Board of Directors or advisory committees. Neuberg:Pharmacyclics: Research Funding; Madrigal Pharmaceuticals: Equity Ownership; Celgene: Research Funding. Davids:Acerta Pharma, Ascentage Pharma, Genentech, MEI pharma, Pharmacyclics, Surface Oncology, TG Therapeutics, Verastem: Research Funding; Research to Practice: Honoraria; AbbVie, Acerta Pharma, Adaptive, Biotechnologies, Astra-Zeneca, Genentech, Gilead Sciences, Janssen, Pharmacyclics, TG therapeutics: Membership on an entity's Board of Directors or advisory committees; AbbVie, Astra-Zeneca, Genentech, Janssen, MEI, Pharmacyclics, Syros Pharmaceuticals, Verastem: Consultancy. Letai:Zeno Pharmaceuticals, Vivid Bioscience, Flash Therapeutics, Dialectic Therapeutics: Membership on an entity's Board of Directors or advisory committees, Other: Cofounder or Advisory Board member; AbbVie, AstraZeneca, Novartis: Consultancy, Research Funding. Wu:Neon Therapeutics: Other: Member, Advisory Board; Pharmacyclics: Research Funding.

Wang: Mission Bio Inc.: Employment. Jacob:Mission Bio Inc.: Employment. Flynn:Mission Bio Inc.: Employment. Ruff:Mission Bio Inc.: Employment. Jones:Mission Bio Inc.: Employment. Neuberg:Pharmacyclics: Research Funding; Madrigal Pharmaceuticals: Equity Ownership; Celgene: Research Funding. Wu:Neon Therapeutics: Other: Member, Advisory Board; Pharmacyclics: Research Funding.

Neuberg: Pharmacyclics: Research Funding; Madrigal Pharmaceuticals: Equity Ownership; Celgene: Research Funding. Wu:Neon Therapeutics: Other: Member, Advisory Board; Pharmacyclics: Research Funding.

Mutation in IKZF3(AIOLOS), a lymphoid transcription factor with a key role in B cell development and function, has been identified as a putative driver of chronic lymphocytic leukemia (CLL) through large-scale WES studies of CLL patients. Prevalent in ~3% CLLs and associated with fludarabine resistance, mutated IKZF3has been detected uniquely as a hotspot mutation (L162R), localized within the DNA binding domain. The functional effects exerted by this mutation on lymphomagenesis remain unexplored, and were the subject of the current study.

Wu: Neon Therapeutics: Equity Ownership.

Large-scale DNA sequencing efforts in chronic lymphocytic leukemia (CLL) have identified a broad array of putative cancer drivers arising from somatic mutations in this disease, but functional understanding of the impact of these genetic events on CLL onset and progression remains to be elucidated. One such example is mutation in the IKZF3gene, encoding the zinc finger protein AIOLOS, mutated in ~2% of CLLs and associated with fludarabine-refractory disease. AIOLOS is a lymphoid-restricted transcription factor and a chromatin remodeler that plays an essential role in B cell development and maturation. In CLL, the IKZF3mutation, also reported in few cases of diffuse large B cell lymphoma and mantle cell lymphoma,targets a highly conserved hotspot (L162R, homologous to murine L161R) that is localized in the 2ndzinc finger of the DNA-binding domain, required for DNA sequence recognition. Given the localization of this hotspot mutation, we hypothesized that it impacts the function of AIOLOS to drive CLL.

Wiestner: Pharmacyclics LLC, an AbbVie Company: Research Funding. Wu:Neon Therapeutics: Equity Ownership.

Collective large-scale sequencing efforts have unexpectedly revealed the high frequency of mutations in the splicing factor genes (SF3B1, U2AF1, SRSF2, ZRSR2) in various solid and hematological cancers, suggesting the association of splicing dysregulation with tumorigenesis. Mutations in SF3B1occur in 5-20% of patients with chronic lymphocytic leukemia (CLL) and are associated with poorer overall survival and chemotherapy resistance. These mutations are restricted to hotspots (>50% at K700E site) and strongly co-occur with ATMmutations (loss-of-function) and deletion of 11q (ATMminimal deleted region). Numerous studies including ours have demonstrated that somatic alterations in this gene cause RNA splicing dysregulation, however, how this splicing factor mutation alone and in combination with ATMdeletion impacts cellular processes and contributes to CLL remains to be fully defined.

Introduction

Introduction: Despite considerable advances in the treatment of chronic lymphocytic leukemia (CLL) with targeted agents including B-cell receptor (BCR) signaling inhibitors, such as the Bruton's tyrosine kinase (BTK) inhibitor ibrutinib and phosphatidylinositol 3-kinase delta (PI3Kδ) inhibitor idelalisib, CLL generally remains incurable. Identifying suitable combination therapies to improve the efficacy of BCR inhibitors may induce deeper remissions and potentially permit treatment discontinuation. Inhibitors of the bromodomain and extra-terminal motif (BET) family of proteins demonstrate preclinical activity against B cell malignancies. Here, we explore the activity of a novel BET inhibitor, GS-5829, in CLL that selectively binds to acetylated lysine recognition motifs in the bromodomains of BET proteins, impeding their interaction with acetylated histones, and ultimately interfering with transcription of BET-regulated target genes.

Despite the remarkable efficacy of novel agents targeting the BTK and BCL2 signaling pathways in CLL, the development of resistance to these drugs has become one of the foremost clinical challenges in the management of CLL patients. An emerging priority is the identification of combinatorial treatments capable of inducing more durable remissions. Recent genomic investigations of CLL have identified altered RNA splicing as a CLL-associated cellular pathway, with heterozygous mutation in the splicing factor SF3B1representing a common lesion in CLL, associated with transcriptomic alterations in numerous CLL survival pathways. We asked whether alternative splicing was also a more general characteristic of CLL (regardless of SF3B1status) over normal B cells, and whether changes in alterations generated by a splicing modulator could provide new opportunities for effective combination therapy.

Faderl: Celgene Corp.: Other: Advisory Board. Wierda:Celgene Corp.: Consultancy; Glaxo-Smith-Kline Inc.: Research Funding. Rezvani:Pharmacyclics: Research Funding. Burger:Pharmacyclics LLC, an AbbVie Company: Research Funding. Keating:Glaxo-Smith-Kline Inc.: Other: Advisory board; Celgene Corp.: Consultancy.

Burger: Boehringer Ingelheim Pharma: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Noxxon: Membership on an entity's Board of Directors or advisory committees; Portola Pharmaceuticals: Research Funding; Gilead: Research Funding; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding. Marasca:Janssen: Honoraria, Other: support for travel to congresses.