Strategy For Chronic Lymphocytic Leukemia Research Articles

Taking the Next Step in Double Refractory Disease: Current and Future Treatment Strategies for Chronic Lymphocytic Leukemia.

Chronic lymphocytic leukemia (CLL) is a monoclonal B-cell lymphoproliferative disease with a high annual incidence in Western countries. As B-cell receptor (BCR) signaling and intrinsic apoptotic resistance play critical roles in the development and survival of CLL cells, therapeutic approaches targeting these pathways have been extensively investigated to tackle this incurable disease. Over the last decade, several Phase 3 trials have confirmed the superior efficacy of covalent Bruton tyrosine kinase inhibitors (cBTKis) and venetoclax, a selective B-cell lymphoma 2 (BCL2) inhibitor, over chemoimmunotherapy. This has been demonstrated in both the treatment-naïve and relapsed/refractory (RR) settings and includes patients with high-risk molecular features. However, these drugs are not curative, with patients continuing to relapse after treatment with both cBTKis and BCL2is, and the optimal treatment strategy for these patients has not been defined. Several novel agents with distinct mechanisms have recently been developed for CLL which have demonstrated efficacy in patients who have previously received cBTKis and BCL2i. In particular, novel BCR-signaling targeting agents have shown promising efficacy in early-phase clinical trials for RR-CLL. Furthermore, cancer immunotherapies such as bispecific antibodies and chimeric antigen receptor T-cells have also shown anti-tumor activity in patients with heavily pretreated RR-CLL. Personalised approaches with these novel agents and combination strategies based on the understanding of resistance mechanisms have the potential to overcome the clinical challenge of what to do next for a patient who has already had a cBTKi and venetoclax.

A CX3CR1 (fractalkine receptor) Small Molecular Antagonist (KAND567) Suppressed the Growth Promoting Effect of Monocytes on Chronic Lymphocytic Leukemia Cells

Background: Chronic lymphocytic leukemia (CLL) is the most prevalent leukemia worldwide. Novel treatment strategies are needed to improve the prognosis. Non-malignant cells of the tumor microenvironment (TME) are of importance for the growth of tumor cells. Monocytes expressing various chemokine receptors as CX3CR1 (the fractalkine receptor (FKNR)) have been shown to promote the growth of tumor cells in vitro and in vivo. In CLL, monocyte derived nurse-like cells (NLC) support the growth of CLL cells, promoting leukemic cell proliferation and survival by secretion of chemokines/cytokines. In this study, we studied the role of autologous monocytes on the survival of CLL cells and the effects of a small molecule antagonist of CX3CR1 (KAND567/FKNRa). KAND567 is in clinical trials for diseases with an activated fractalkine system as part of the pathobiology including covid-19 infected patients as well as patients with acute myocardial infarction and ovarian carcinoma. Methods: Primary CLL cells and B cells (CD19 +) of healthy donors as well as autologous monocytes (CD14 +) from CLL patients (n=22) and healthy control donors (n=11) were cultured alone or in co-cultures (isolated B cells together with autologous monocytes) with the FKNRa (250 and 1000 nM) for up to 120 h. Apoptosis of CD19 + cells and monocytes was analysed by Annexin V/PI staining, Western blotting and the IncuCyte® Live Cell Imaging System. The NBT assay was also applied to check morphological changes of monocytes. Plasma concentration of fractalkine (CX3CL1) was determined by ELISA. Results and discussion: CLL patients had a significant (p=0.0001) increase in the plasma concentration of CX3CL1 compared to healthy controls. CLL cells as well as normal B cells did not express surface CX3CR1. However, monocytes from both CLL patients and healthy donors expressed high levels of surface CX3CR1. A significantly higher proportion of pro-inflammatory intermediate (CD14 +/CD16 +) and non-classical (CD14 -/CD16 +) monocytes (p<0.0001 and <0.01 resp.) as well as intermediate and non-classical CX3CR1 + monocytes were observed in patients with active CLL compared to healthy donors (p<0.0001 and <0.01 resp.). Moreover, CX3CR1 + intermediate and non-classical monocytes were significantly higher in CLL patients with active disease compared to those with early-stage disease (p<0.0001 and <0.001 resp.). A growth supportive effect of autologous monocytes was observed on survival of both CLL cells and healthy donor B cells in 120 h cell co-culture experiments (p<0.001 and <0.01 resp.). FKNRa reduced the number of alive CLL cells in a dose-dependent manner in co-cultures with autologous monocytes (p<0.05). No effect was seen on CD19 + B cells of healthy donors co-cultured with autologous monocytes. FKNRa did not induce apoptosis of CLL cells alone. Furthermore, FKNRa also inhibited transformation of CLL monocytes to NLC (p<0.05) which was not the case for normal monocytes. Conclusion: The CX3CR1/CX3CL1 (fractalkine receptor/ligand)) axis appears to be activated in CLLand might be of importance for the NLC-driven growth of CLL cells. This is the first report of a small molecule antagonist of CX3CR1 reducing the growth supportive effect of autologous CLL monocytes on leukemic CLL cells. Disruption of growth promotion induced by monocytes may prevent leukemic cell proliferation and survival. Inhibition of autologous monocytes in the TME might represent a new therapeutic strategy in CLL, complementary to treatment with drugs that directly target the tumor cells.

Cost Effectiveness of Molecular Diagnostic Testing Algorithms for the Treatment Selection of Frontline Ibrutinib for Patients with Chronic Lymphocytic Leukemia in Australia.

Clinical indications for ibrutinib reimbursement in Australia should consider the inclusion of patients with chronic lymphocytic leukemia (CLL) harboring prognostically unfavorable TP53/IGHV genomic aberrations. This study assessed the cost effectiveness of five first-line treatment strategies in CLL for young (aged ≤65years), fit patients without significant comorbidities: (1) no testing (fludarabine, cyclophosphamide and rituximab [FCR] for all), (2) test for del(17p) only, (3) test for TP53 gene mutation status, (4) test for TP53 and IGHV gene mutation status and (5) no testing (ibrutinib for all). A decision analytic model (decision tree and partitioned survival model) was developed from the Australian healthcare system perspective with a lifetime horizon. Comparative treatment effects were estimated from indirect treatment comparisons and survival analysis using several studies. Costs, utility and adverse events were derived from public literature sources. Deterministic and probabilistic sensitivity analyses explored the impact of modeling uncertainties on outcomes. Strategy 1 was associated with 5.69 quality-adjusted life-years (QALYs) and cost 458,836 Australian dollars (AUD). All other strategies had greater effectiveness but were more expensive than Strategy 1. At the willingness-to-pay (WTP) threshold of 100,000 AUD per QALY gained, Strategy 1 was most cost effective with an estimated probability of 68.8%. Strategy 4 was cost effective between thresholds 155,000-432,300 AUD per QALY gained, and Strategy 5 >432,300 AUD per QALY gained. Population targeting using mutation testing for TP53 and IGHV when performed with del(17p) testing specifically in the context of frontline ibrutinib choice does not make a cost-ineffective treatment into a cost-effective treatment.

SF3B1 mutation-mediated sensitization to H3B-8800 splicing inhibitor in chronic lymphocytic leukemia.

Splicing factor 3B subunit 1 (SF3B1) is involved in pre-mRNA branch site recognition and is the target of antitumor-splicing inhibitors. Mutations in SF3B1 are observed in 15% of patients with chronic lymphocytic leukemia (CLL) and are associated with poor prognosis, but their pathogenic mechanisms remain poorly understood. Using deep RNA-sequencing data from 298 CLL tumor samples and isogenic SF3B1 WT and K700E-mutated CLL cell lines, we characterize targets and pre-mRNA sequence features associated with the selection of cryptic 3' splice sites upon SF3B1 mutation, including an event in the MAP3K7 gene relevant for activation of NF-κB signaling. Using the H3B-8800 splicing modulator, we show, for the first time in CLL, cytotoxic effects in vitro in primary CLL samples and in SF3B1-mutated isogenic CLL cell lines, accompanied by major splicing changes and delayed leukemic infiltration in a CLL xenotransplant mouse model. H3B-8800 displayed preferential lethality towards SF3B1-mutated cells and synergism with the BCL2 inhibitor venetoclax, supporting the potential use of SF3B1 inhibitors as a novel therapeutic strategy in CLL.

Inhibition of MYC translation through targeting of the newly identified PHB-eIF4F complex as a therapeutic strategy in CLL

AbstractDysregulation of messenger RNA (mRNA) translation, including preferential translation of mRNA with complex 5′ untranslated regions such as the MYC oncogene, is recognized as an important mechanism in cancer. Here, we show that both human and murine chronic lymphocytic leukemia (CLL) cells display a high translation rate, which is inhibited by the synthetic flavagline FL3, a prohibitin (PHB)-binding drug. A multiomics analysis performed in samples from patients with CLL and cell lines treated with FL3 revealed the decreased translation of the MYC oncogene and of proteins involved in cell cycle and metabolism. Furthermore, inhibiting translation induced a proliferation arrest and a rewiring of MYC-driven metabolism. Interestingly, contrary to other models, the RAS-RAF-(PHBs)-MAPK pathway is neither impaired by FL3 nor implicated in translation regulation in CLL cells. Here, we rather show that PHBs are directly associated with the eukaryotic initiation factor (eIF)4F translation complex and are targeted by FL3. Knockdown of PHBs resembled FL3 treatment. Importantly, inhibition of translation controlled CLL development in vivo, either alone or combined with immunotherapy. Finally, high expression of translation initiation–related genes and PHBs genes correlated with poor survival and unfavorable clinical parameters in patients with CLL. Overall, we demonstrated that translation inhibition is a valuable strategy to control CLL development by blocking the translation of several oncogenic pathways including MYC. We also unraveled a new and direct role of PHBs in translation initiation, thus creating new therapeutic opportunities for patients with CLL.

NOTCH1 Signaling Is Dysregulated in CLL By the Deubiquitinase USP28 That Is Recurrently Affected By Del(11q) and Can be Therapeutically Targeted

Active NOTCH1 signaling is present in approximately half of chronic lymphocytic leukemia (CLL) patients and was shown to be a marker for disease outcome. The NOTCH1 signaling pathway can be aberrantly activated through mutations in the NOTCH1 gene itself or co-factors such as FBXW7, resulting in protein stabilization of cleaved NOTCH1 (NICD). However, 50% of CLL cases with NOTCH1 activation do not harbor alterations in these genes, suggesting additional mechanisms for NOTCH1 hyperactivation. Since NICD can be stabilized due to defects in the ubiquitination/degradation machinery, we aimed to elucidate the role of the on chromosome 11q23 located deubiquitinase USP28 in the regulation of NOTCH1 activity and as a novel therapeutic target for NOTCH1-dependent CLL patients. First, we evaluated USP28 expression levels in a previously published cohort of 285 untreated CLL patients characterized via SNP array analysis and gene expression profiling (Edelmann et al, Blood 2012; Bloehdorn et al, Nat Commun 2021). The presence of monoallelic USP28 deletion was identified in 90% of CLL patients harboring a heterozygous deletion of chromosome 11q (del(11q); n=96), resulting in a significant decrease of USP28 RNA expression in comparison to non-del(11q) cases (n=199, P<0.0001). In non-del(11q) cases, USP28 expression levels were variable and assessment of protein levels of USP28 and its target proteins by western blotting in primary CLL cells (n=8) revealed a correlation between high protein levels of USP28 and remarkably high levels of NICD and other USP28 targets such as c-MYC or c-JUN. In addition, interaction of USP28 with NICD was shown by immunoprecipitation assays. In parallel, we implemented the CRISPR/Cas9 system to generate HG3 CLL-derived cell lines harboring USP28 biallelic deletion (HG3-USP28KO, n=5), monoallelic 11q deletion (HG3-del(11q), 17 Mb size including both ATM and USP28 genes, n=3), biallelic ATM truncation (HG3-ATMKO, n=3) or the combination of both events (HG3-del(11q) ATMKO, n=3). Overexpression of USP28 in HG3WT and HG3-USP28KO cells increased NOTCH1 activity measured by luciferase reporter assays (P<0.05, P<0.001, respectively). In contrast, in HG3-del(11q) cells decreased USP28 RNA and protein expression resulted in reduced NOTCH1 activity compared to HG3WT cells (P<0.05). Notably, HG3-del(11q) ATMKO cell lines showed even lower levels of NOTCH1 activity (P<0.01). Given that ATM phosphorylates USP28 (Zhang et al, Cell 2006), we hypothesized that in CLL cells ATM regulates USP28 activity and thereby also downstream NOTCH1 signaling. Indeed, NOTCH1 activity was reduced in HG3-ATMKO cells (P<0.01). However, USP28 overexpression in HG3-ATMKO cells did not lead to increased NOTCH1 signaling activity as observed in HG3WT cells. Moreover, pharmacological ATM inhibition (5 µM of KU-55933) also resulted in reduced NOTCH1 activity in HG3WT cells (P<0.0001) and reduced NICD levels in primary CLL cells (n=12; P<0.05). We finally explored the impact of the small molecule USP28 inhibitor AZ1 (alone or in combination with ibrutinib or venetoclax) on NICD protein levels and viability of primary CLL cells (n=15). AZ1 treatment (10 µM) resulted in decreased NICD levels, independent of mutations within NOTCH1 or FBXW7 (P<0.01). ATP quantification and 7-AAD staining revealed that increasing doses of AZ1 significantly reduced viability of NOTCH1/FBXW7-MUT primary cells (n=5) as well as of NOTCH1/FBXW7-WT cells (n=6) expressing high protein levels of USP28 and NICD (P<0.01; P<0.05, respectively). In contrast, AZ1 treatment did not affect viability of CLL patient cells expressing low NICD levels (n=4; P=0.56) Furthermore, AZ1 combined with venetoclax potentiated the reduction of cell viability especially in NOTCH1/FBXW7-MUT or patient cells expressing high USP28 and NICD levels (P<0.001; P<0.05). We propose that high levels of the deubiquitinase USP28 are responsible for hyperactivation of NOTCH1 signaling in CLL. This hypothesis is further confirmed in del(11q) CLL cells, where USP28 heterozygous deletion resulted in low NOTCH1 activity. In addition, our data reveal that USP28 activity is regulated by ATM, uncovering a novel mechanism of NOTCH1 modulation via the ATM-USP28-NOTCH1 axis (Figure 1). Finally, we demonstrate that USP28 inhibition by AZ1 suppresses USP28-mediated NOTCH1 activation and could represent a novel therapeutic strategy in CLL. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Ezrin Is a Potential Druggable Target in Chronic Lymphocytic Leukemia

Despite development in therapeutic strategies for chronic lymphocytic leukemia (CLL), most patients remain incurable, relapse, or are refractory to current treatments, indicating the need to expand the antineoplastic repertoire for this disease. Ezrin (EZR) is a known oncogene in solid tumors, but its role in hematological neoplasms is still poorly explored. There is evidence that EZR plays a key function in cell survival and activation of BCR-mediated signaling in B-cell lymphomas. Here, we uncover the expression of EZR in samples from CLL and healthy donors, and the cellular and molecular effects of the NSC305787, a pharmacological EZR inhibitor, in CLL cellular models. For gene expression studies, EZR mRNA expression data from healthy donors (normal lymphocytes, n = 11) and CLL patients (n = 103) samples were derived from the publicly accessible AmaZonia! database 2008 (discovery cohort). Purified CD19+ cells from peripheral blood samples were collected from 56 CLL patients and 10 age-matched healthy donors from the University Hospital of the Medical School of Ribeirão Preto (validation cohort). For functional and molecular assays, peripheral blood mononuclear cells of 24 CLL patients from the Instituto do Câncer do Estado de São Paulo were used. All samples were obtained after informed consent and protocol approved by the Ethical Committee of the Institution. For functional transcriptomics, RNA-seq data published by Landau et al. were obtained from cBioPortal, and all transcripts were pre-ranked according to their differential expression by comparing samples with high versus low EZR expression using limma-voom package in Galaxy and used for gene set enrichment analysis (GSEA). The MEC-1 cell line was cultured following the recommendations of DSMZ. PBMCs from CLL patients were cultured in RPMI Media 1640 and 10% autologous serum. NSC305787 was used as an EZR inhibitor. Cell viability was determined by MTT assay, apoptosis rates were determined by annexin V/PI labeling and flow cytometry, DNA content was performed by PI labeling and flow cytometry, protein expression was determined by Western Blot assay, and gene expression was evaluated by quantitative PCR. Statistical analyses were performed using GraphPad Prism 8; Mann-Whitney test, Student's t-test, or ANOVA and Bonferroni post-test were used for measurable factors, as appropriate. A value of p < 0.05 was considered statistically significant. EZR was highly expressed in lymphocytes derived from CLL patients (discovery cohort: p = 0.003; validation cohort: p = 0.005). GSEA indicated that high EZR expression was positively associated with relevant signaling pathways associated with CLL development and progression, including p53, PI3K/AKT/mTOR, NFkB, and MAPK (all p < 0.05 and FDR q < 0.05). Of note, pharmacological inhibition of EZR had remarkable antineoplastic effects in primary cells derived from CLL patients, corroborating the potential relevance of this target in disease maintenance. In a CLL cell line, EZR inhibition reduced viability, clonogenicity, cell cycle progression, and induced apoptosis (all p < 0.05). Pharmacological EZR inhibition also reduced activation of the ERK, S6RP, and NFkB, indicating that EZR not only associates, but participates in the activation of these signaling pathways in CLL. In conclusion, our results indicate that EZR is high expressed in CLL and associated with molecular signatures relevant to the development and maintenance of the disease. Pharmacological EZR inhibitions attenuate various cellular and molecular behaviors associated with high risk in CLL and may present a new class of drugs to expand the treatment repertoire of this disease. Supported by: FAPESP, CNPQ, and CAPES.

Ezrin is highly expressed and a druggable target in chronic lymphocytic leukemia

AimsDespite the development of therapeutic strategies for chronic lymphocytic leukemia (CLL), most patients remain incurable, relapse, or refractory to current treatments, indicating the need to expand the antineoplastic repertoire for this disease. Ezrin (EZR) is a known oncogene in solid tumors and plays a key role in cell survival and BCR-mediated signaling activation in B-cell lymphomas. However, its role in hematological neoplasms remains poorly explored. Main methodsThe present study assessed EZR expression in samples from CLL patients and healthy donors and evaluated the cellular and molecular effects of a pharmacological EZR inhibitor, NSC305787, in CLL cellular models. Key findingsEZR was highly expressed and positively associated with relevant signaling pathways related to CLL development and progression, including TP53, PI3K/AKT/mTOR, NF-κB, and MAPK. NSC305787 reduced viability, clonogenicity, and cell cycle progression and induced apoptosis in CLL cells. Pharmacological EZR inhibition also attenuated ERK, S6RP, and NF-κB activation, indicating that EZR not only associates with but also activates these signaling pathways in CLL. Ex vivo assays revealed that the EZR inhibition-induced cell viability reduction was independent of molecular risk and the Binet stage. SignificanceOur study provides insights into EZR as a pharmacological target in CLL, shedding light on a novel strategy for treating this disease.

Disruption of Nurse-like Cell Differentiation as a Therapeutic Strategy for Chronic Lymphocytic Leukemia.

Chronic lymphocytic leukemia (CLL) is the most common adult leukemia, but, despite advances in treatment, many patients still experience relapse. CLL cells depend on interactions with supportive cells, and nurse-like cells (NLCs) are the major such cell type. However, little is known about how NLCs develop. Here, we performed DNA methylation analysis of CLL patient-derived NLCs using the 850K Illumina array, comparing CD14+ cells at day 1 (monocytes) versus day 14 (NLCs). We found a strong loss of methylation in AP-1 transcription factor binding sites, which may be driven by MAPK signaling. Testing of individual MAPK pathways (MEK, p38, and JNK) revealed a strong dependence on MEK/ERK for NLC development, because treatment of patient samples with the MEK inhibitor trametinib dramatically reduced NLC development in vitro. Using the adoptive transfer Eµ-TCL1 mouse model of CLL, we found that MEK inhibition slowed CLL progression, leading to lower WBC counts and to significantly longer survival time. There were also lower numbers of mouse macrophages, particularly within the M2-like population. In summary, NLC development depends on MEK signaling, and inhibition of MEK leads to increased survival time in vivo. Hence, targeting the MEK/ERK pathway may be an effective treatment strategy for CLL.

Abstract LB050: Targeting cells in the microenvironment as a novel therapeutic strategy for chronic lymphocytic leukemia

Abstract Chronic lymphocytic leukemia (CLL) is a disease that affects the elderly, characterized by the accumulation of mature B cells in the bloodstream. With an estimation incidence of 21,250 new cases diagnosed and 4,320 deaths for 2021, CLL is still considered a non-curable disease despite the wide therapeutic alternatives. CLL, like other malignant diseases, involves the activity of different cells in the microenvironment that support the leukemic cells by providing survival signals, even in presence of therapeutic agents. Among these cells, nurse-like cells (NLC) are of great importance. NLCs are M2-like macrophages known to be developed from blood monocytes, which need close contact with CLL cells for both their differentiation and their protective function. They provide multiple survival signals to CLL cells, and protect them against agents like the BTK inhibitor ibrutinib. Despite their relevance, little research has been done to elucidate the program that drives NLC differentiation. Here, we addressed this question by looking at the DNA methylation signatures before and after NLC differentiation. We found that NLC differentiation produced a marked DNA hypomethylation when compared to freshly isolated monocytes, characterized by the enrichment in AP-1 transcription binding sites. AP-1 is known to be regulated by MAP kinases, so we decided to chemically inhibit these pathways and found that specific MEK inhibition lead to reduced numbers of NLCs in vitro. Further studies showed that indeed NLCs have an activated MEK signaling as seen by the basal phosphorylation of ERK in vitro by confocal microscopy. We then used the adoptive transfer Eµ-TCL1 mice model to seek the effect of MEK inhibition in vivo. By treating the mice with trametinib, an FDA-approved MEK inhibitor, we found an increase in mouse survival when compared with the vehicle control. In addition, we observed a reduced number of monocyte/macrophage populations, especially in those expressing EGR2, a known M2 marker, suggesting that MEK inhibition causes a disruption in CLL-supportive macrophages both in vitro and in vivo. In conclusion, we observed that NLC differentiation strongly depends on the MEK signaling pathway, and that its inhibition leads to reduced myeloid supportive cells for CLL cells, followed by a reduced leukemic progression and increased CLL survival in vivo. Thus, we propose that MEK inhibition could be a potential therapeutic alternative for CLL. Further research is ongoing to determine how MEK signaling is activated during NLC differentiation. Citation Format: Giovanna Merchand Reyes, Ramasamy Santhanam, Frank H. Robledo Avila, Christoph Weigel, Juan D. Ruiz Rosado, Xiaokui Mo, Santiago Partida Sanchez, Jennifer A. Woyach, Christopher C. Oakes, Susheela Tridandapani, Jonathan P. Butchar. Targeting cells in the microenvironment as a novel therapeutic strategy for chronic lymphocytic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB050.

Lenalidomide enhances CD23.CAR T cell therapy in chronic lymphocytic leukemia

Chimeric antigen receptors (CAR)-modified T cells are an emerging therapeutic tool for chronic lymphocytic leukemia (CLL). However, in patients with CLL, well-known T-cell defects and the inhibitory properties of the tumor microenvironment (TME) hinder the efficacy of CAR T cells. We explored a novel approach combining CARs with lenalidomide, an immunomodulatory drug that tempers the immunosuppressive activity of the CLL TME. T cells from patients with CLL were engineered to express a CAR specific for CD23, a promising target antigen. Lenalidomide maintained the in vitro effector functions of CD23.CAR+ T cells effector functions in terms of antigen-specific cytotoxicity, cytokine release and proliferation. Overall, lenalidomide preserved functional CAR T-CLL cell immune synapses. In a Rag2−/−γc −/−-based xenograft model of CLL, we demonstrated that, when combined with low-dose lenalidomide, CD23.CAR+ T cells efficiently migrated to leukemic sites and delayed disease progression when compared to CD23.CAR+ T cells given with rhIL-2. These observations underline the therapeutic potential of this novel CAR-based combination strategy in CLL.

Chromothripsis in Chronic Lymphocytic Leukemia: A Driving Force of Genome Instability.

Chromothripsis represents a mechanism of massive chromosome shattering and reassembly leading to the formation of derivative chromosomes with abnormal functions and expression. It has been observed in many cancer types, importantly, including chronic lymphocytic leukemia (CLL). Due to the associated chromosomal rearrangements, it has a significant impact on the pathophysiology of the disease. Recent studies have suggested that chromothripsis may be more common than initially inferred, especially in CLL cases with adverse clinical outcome. Here, we review the main features of chromothripsis, the challenges of its assessment, and the potential benefit of its detection. We summarize recent findings of chromothripsis occurrence across hematological malignancies and address its causes and consequences in the context of CLL clinical features, as well as chromothripsis-related molecular abnormalities described in published CLL studies. Furthermore, we discuss the use of the current knowledge about genome functions associated with chromothripsis in the optimization of treatment strategies in CLL.

Activation of Interferon Signaling in Chronic Lymphocytic Leukemia Cells Contributes to Apoptosis Resistance via a JAK-Src/STAT3/Mcl-1 Signaling Pathway

Besides their antiviral and immunomodulatory functions, type I (α/β) and II (γ) interferons (IFNs) exhibit either beneficial or detrimental effects on tumor progression. Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of abnormal CD5+ B lymphocytes that escape death. Drug resistance and disease relapse still occur in CLL. The triggering of IFN receptors is believed to be involved in the survival of CLL cells, but the underlying molecular mechanisms are not yet characterized. We show here that both type I and II IFNs promote the survival of primary CLL cells by counteracting the mitochondrial (intrinsic) apoptosis pathway. The survival process was associated with the upregulation of signal transducer and activator of transcription-3 (STAT3) and its target anti-apoptotic Mcl-1. Furthermore, the blockade of the STAT3/Mcl-1 pathway by pharmacological inhibitors against STAT3, TYK2 (for type I IFN) or JAK2 (for type II IFN) markedly reduced IFN-mediated CLL cell survival. Similarly, the selective Src family kinase inhibitor PP2 notably blocked IFN-mediated CLL cell survival by downregulating the protein levels of STAT3 and Mcl-1. Our work reveals a novel mechanism of resistance to apoptosis promoted by IFNs in CLL cells, whereby JAKs (TYK2, JAK2) and Src kinases activate in concert a STAT3/Mcl-1 signaling pathway. In view of current clinical developments of potent STAT3 and Mcl-1 inhibitors, a combination of conventional treatments with these inhibitors might thus constitute a new therapeutic strategy in CLL.

Phosphoinositide 3-Kinase Signaling in the Tumor Microenvironment: What Do We Need to Consider When Treating Chronic Lymphocytic Leukemia With PI3K Inhibitors?

Phosphoinositide 3-kinases (PI3Ks) and their downstream proteins constitute a signaling pathway that is involved in both normal cell growth and malignant transformation of cells. Under physiological conditions, PI3K signaling regulates various cellular functions such as apoptosis, survival, proliferation, and growth, depending on the extracellular signals. A deterioration of these extracellular signals caused by mutational damage in oncogenes or growth factor receptors may result in hyperactivation of this signaling cascade, which is recognized as a hallmark of cancer. Although higher activation of PI3K pathway is common in many types of cancer, it has been therapeutically targeted for the first time in chronic lymphocytic leukemia (CLL), demonstrating its significance in B-cell receptor (BCR) signaling and malignant B-cell expansion. The biological activity of the PI3K pathway is not only limited to cancer cells but is also crucial for many components of the tumor microenvironment, as PI3K signaling regulates cytokine responses, and ensures the development and function of immune cells. Therefore, the success or failure of the PI3K inhibition is strongly related to microenvironmental stimuli. In this review, we outline the impacts of PI3K inhibition on the tumor microenvironment with a specific focus on CLL. Acknowledging the effects of PI3K inhibitor-based therapies on the tumor microenvironment in CLL can serve as a rationale for improved drug development, explain treatment-associated adverse events, and suggest novel combinatory treatment strategies in CLL.

High Clonal Complexity of Resistance Mechanisms Occurring at Progression after Single-Agent Targeted Therapy Strategies in Chronic Lymphocytic Leukemia

Progression of chronic lymphocytic leukemia (CLL) on venetoclax (VEN) and BTK inhibitors (BTKi) is associated with acquired genomic variants in BCL2/MCL1/BCL2L1 and BTK/PLCG2, respectively, in some patients. We aimed to assess the clonal structure and evolution of resistance in patients (pts) with progressive disease treated with single agent VEN or BTKi (or both as sequential monotherapies) using next generation sequencing (NGS) and single cell sequencing. Seven pts with CLL and 1 with mantle cell lymphoma (MCL) with disease progression on VEN, ibrutinib (IBR) or zanubrutinib (ZANU) were identified from patients treated at our institutions. Pts were selected on the basis of multiple known resistance mechanisms from previous analysis of mutations (muts) and copy number changes detected using clinical bulk NGS targeting genes of interest including BCL2, MCL1, BCL2L1, BAX, BAK1, BTK, PLCG2, CXCR4, as well as TP53 and SF3B1. Of the 8 pts selected for single cell analysis, all had disease that was relapsed/refractory to chemotherapy prior to receiving either VEN (3 pts), BTKi (2 pts) or sequential VEN-BTKi (3 pts). 6,520-16,378 individual cells from 9 samples (8 pts) were analyzed (total 103,388 cells) using a custom panel targeting pt-specific muts on the Tapestri platform (Mission Bio). A summary of genomic abnormalities detected across the cohort is presented in Figure 1. We first evaluated the relationship between genomic resistance mechanisms within the context of single agent (VEN or BTKi) as well as sequential VEN-BTKi treatment. In CLL pts treated with a single agent, all BCL2 muts in VEN pts and BTK muts in IBR or ZANU pts were identified in different subclones consistent with an oligoclonal pattern of disease progression with independent clonal acquisition of resistance mechanisms. Both pts who received ZANU (either as a single agent or sequentially) harbored the BTK L528W mut (previously described as enriched in ZANU progressors; Handunnetti ASH 2019) in independent clones from BTK C481 muts. In pts who received sequential VEN-BTKi treatment, clones were observed that harbored established or novel dual genomic resistance mechanisms within the same cell (BTK mut/MCL1 amp in CLL, BTK/BAX muts in MCL). However, this was not observed in all clones or for all pts, suggesting the presence of further undetected resistance mechanisms (genetic or other). Given the unique ability of single cell sequencing to resolve mut context within a clonal hierarchy, we next assessed this phenomenon within our cohort utilizing other muts known to be present in these tumors. Analysis of TP53 muts exemplified the diversity of clonal patterns observed, with resistance muts being detected subclonally to parental TP53 muts in some pts and independently of TP53 muts in others. In addition, further evolution of resistant clones was observed through the development of TP53 muts within clones harboring acquired resistance muts, consistent with continued clonal evolution within the resistant disease compartment. In one pt, post-resistance clonal evolution was identified through the clonal acquisition of a CXCR4 mut within a BTK mutated population. Finally, to understand the contribution of BTK zygosity and gender to BTKi resistance (given its location on the X-chromosome), we performed single cell analysis on a disease specimen from a female pt with progressive MCL harboring multiple BTK mutations following treatment with sequential VEN-BTKi. Analysis revealed four clonally independent heterozygous BTK muts inferring the sufficiency of a single mutant allele to drive resistance in this context. Interestingly, this pt also harbored a BCL2 mut and a BAX mut, the latter co-occurring with a BTK mut (BCL2 not assessable). This pt therefore represents the first description of BCL2 or BAX muts occurring in a pt with progressive MCL on VEN and the first of a BTK L528W mut in MCL progressing on ZANU. In summary, these data highlight the significant clonal complexity of CLL progression on VEN and BTKi. Our data show that disease progression in this context is consistently oligoclonal with separate clones harboring distinct identifiable resistance mechanisms. These data have pt-specific implications for the potential utility of cycling back to previously efficacious targeted therapies as well as providing a strong rationale for the early use of disease-appropriate combination targeted therapies. Disclosures Anderson: Walter and Eliza Hall Institute: Patents &amp; Royalties: milestone and royalty payments related to venetoclax.. Handunnetti:AbbVie: Other: Travel expenses; Roche: Honoraria; Gilead: Honoraria. Yeh:Novartis: Honoraria; Gilead: Research Funding. Tam:BeiGene: Honoraria; Janssen: Honoraria, Research Funding; AbbVie: Honoraria, Research Funding. Seymour:Morphosys: Consultancy, Honoraria; Mei Pharma: Consultancy, Honoraria; Gilead: Consultancy; AstraZeneca: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Honoraria, Research Funding; F. Hoffmann-La Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Research Funding; AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Nurix: Honoraria. Roberts:Janssen: Research Funding; Servier: Research Funding; AbbVie: Research Funding; Genentech: Patents &amp; Royalties: for venetoclax to one of my employers (Walter &amp; Eliza Hall Institute); I receive a share of these royalties. Blombery:Amgen: Consultancy; Novartis: Consultancy; Invivoscribe: Honoraria; Janssen: Honoraria.

Elucidating nurse-like cell development as a new therapeutic strategy for chronic lymphocytic leukemia

Abstract Chronic lymphocytic leukemia (CLL) is the most common adult leukemia, with the NIH estimating 20,720 new cases and 3,930 deaths in 2019. Despite advances in treatment, some patients still relapse (eg. up to 10 to 18% of ibrutinib-treated patients). CLL cells depend on interactions with supportive cells for survival, and nurse-like cells (NLCs) are the major such cell type. However, little is known about how NLCs develop. Thus, our objective is to elucidate NLC development for the purpose of identifying novel therapeutic targets that may aid in the treatment of CLL. We examined DNA methylation patterns of NLCs and found unusually strong MAPK and AP-1 transcription factor signatures. qPCR showed upregulation of MAF, MAFF and JUN in NLCs, supporting this epigenetics analysis. To test the role of the high MAPK signature we treated NLC cultures with inhibitors of Mek, p38 or Jnk. Results showed that Mek inhibition dramatically reduced NLC development. Of note, we did not detect phosphorylated Erk in CLL cells within treated or control cultures, suggesting that the Mek inhibitor acted directly against the CD14+-derived cells. We then tested Mek inhibition in vivo with the Eμ-TCL1 mouse model of CLL and found that it led to significantly longer survival time and lower white-blood-cell counts. In conclusion, we found that NLC development is dependent on Mek signaling and that Mek inhibition extends survival in vivo. This suggests that targeting the Mek/Erk pathway may be an effective treatment strategy for CLL.

[Treatment strategies for chronic lymphocytic leukemia in the era of novel targeted therapies].

Molecular targeted therapies with small molecule inhibitors and antibodies have rapidly replaced chemoimmunotherapy, which has been the gold standard of care for patients with chronic lymphocytic leukemia (CLL). We discuss the current treatment strategies for CLL with special emphasis on genomic and molecular risk factors including IGHV unmutated status, 11q deletion, and 17p deletion. Ibrutinib and venetoclax are two molecular targeted agents currently available in Japan. They are highly effective, well tolerated, and have improved overall survival. Therefore, molecular targeted therapies are preferred to chemoimmunotherapy for most patients. Ongoing studies will clarify the optimal option between combination and sequence of treatment regimens with an appropriate timing of therapeutic intervention for longer survival. We are nearing an era of chemotherapy-free CLL management.

BTK inhibitors and anti-CD20 monoclonal antibodies for treatment-naïve elderly patients with CLL.

Older patients account for the majority of patients with chronic lymphocytic leukemia (CLL), and so strategies for managing CLL in this population is of upmost importance. Inhibition of Bruton’s tyrosine kinase (BTK) has been a successful therapeutic strategy in CLL, and the first-generation BTK inhibitor ibrutinib has been shown to be superior to standard chemoimmunotherapy in multiple studies specifically targeting older patients. A second-generation BTK inhibitor, acalabrutinib, has also been studied in CLL, and has recently been granted breakthrough designation by the United States Food and Drug Administration. One ongoing question is whether the addition of anti-CD20 monoclonal antibodies improve response or response durability with BTK inhibitors. In this review, we will discuss clinical trials of ibrutinib and acalabrutinib in older patients with CLL, and the possible contributions of anti-CD20 antibodies to these therapies.

Natural Killer Cell Hypo-responsiveness in Chronic Lymphocytic Leukemia can be Circumvented In Vitro by Adequate Activating Signaling.

Chronic lymphocytic leukemia (CLL) is characterized by an acquired immune dysfunction, which may underlie the hampered efficacy of cellular immunotherapy. Most data on dampened immune responses in CLL come from studies investigating CLL and T cell interactions. Natural killer (NK) cells may be an attractive alternative source of effector cells in immunotherapy in CLL, provided that functionality is retained within the CLL micro-environment. Despite their important role in anti-tumor responses, NK cells are not extensively characterized in CLL. Here, we studied the expression of activating and inhibitory receptors on CLL-derived and healthy control (HC) NK cells, and their functional response towards several stimuli.NK cells from CLL patients have an increased maturation stage, with an expansion of NKG2C+ NK cells in CMV seropositive individuals. The cytotoxicity receptor NKG2D is downregulated, and the killing capacity through this receptor was markedly reduced in CLL-derived NK cells. In contrast, activation via CD16 (FCγRIII) led to adequate activation and functional responses in CLL-derived NK cells. These findings indicate that NK cells in CLL are not intrinsically defect and still perform effector functions upon adequate activating signaling. Clinical relevance of this finding was shown by treatment with novel nanobody-Fc constructs, which induced cytotoxic responses in both CLL- and HC-derived NK cells via CD16. Our results show that NK cells, in contrast to the T cell compartment, retain their function within the CLL micro-environment, provided that they receive an adequate activating signal. These findings warrant future studies on NK cell mediated immunotherapeutic strategies in CLL.

Preliminary Results of Ibrutinib Followed By Ofatumumab Consolidation in Previously Untreated Patients with Chronic Lymphocytic Leukemia (CLL): GELLC7 Trials from the Spanish Group of CLL (GELLC)

Introduction. Despite the high proportion of prolonged remissions obtained with ibrutinib in patients with CLL, complete responses (CR) are rarely observed. For the purpose of increasing the deepness of response, ibrutinib has been tested in combination with other drugs that exert a different mechanism of action. Thus, monoclonal antibodies (mAbs) have been concomitantly combined with ibrutinib in untreated or R/R CLLs. Nonetheless, several data derived from both in vitro and clinical studies do not support a synergistic effect of the concomitant administration of ibrutinib with anti-CD20 mAbs. Herein, we present the preliminary results of a multi-center, non-randomized phase 2 study aimed to determine the efficacy and safety of the sequential treatment of CLL patients with ibrutinib followed, in those not attaining CR, by a consolidation phase with ofatumumab (GELLC-7, EudraCT number 2016-004937-26). Patients and methods. Patients aged ≥18 years, physically fit (CIRS score &lt; 6) with treatment-naïve CLL were enrolled in this study. Patients received an induction phase consisting of 12 cycles (28-day) of ibrutinib in monotherapy at 420 mg once daily. Patients attaining a CR after this induction phase were kept on ibrutinib until progression. In contrast, patients not obtaining a CR also continued on ibrutinib but received a consolidation treatment with 7 doses of ofatumumab (300 mg D1 and 1000mg D8 of C13, 1000 mg D1 of C14-C18). The primary endpoint of the study was the CR rate assessed after 20 cycles of treatment (2 months after completing ofatumumab consolidation). Results. 84 patients with a median age 69 years (range 38-84 yrs), 71% male, were included in this study. At inclusion, 83.3% had Binet stage B/C, 61% unmutated IGHV status, and 19% high risk genetic aberrations (7.6% 17p deletion and/or TP53mut, and 11.4% 11q deletion). At the interim data cut-off (June 2019), 7 patients had discontinued the study (progression to Richter transformation, n=1; patient withdrawal, n=3; adverse events [AE], n=3, including one G5 AE), 5 of them during the first 12 cycles of treatment. Sixty-seven patients received the induction phase with 12 cycles of ibrutinib, whereas 22 patients completed 20 cycles of treatment and were evaluable for the primary endpoint of the study. After 12 cycles of ibrutinib, 3 patients (4.5%) were in CR, 54 patients (80.5%) in PR, 6 patients (9%) in PR with lymphocytosis, and 4 patients (6%) in SD. In 20 patients receiving the consolidation with ofatumumab an improvement in response was observed, with 8/20 patients (40%) attaining a CR (7 patients converted PR to CR, and one patient SD to CR), whereas the remaining 12 patients were classified as PR. Two patients that were already in CR at cycle 12 maintained the CR under ibrutinib monotherapy. MRD was undetectable in blood (&lt;10-4 by flow cytometry) only in one of the 22 patients. With a median follow-up of 15 months (2 - 20.2 months), the estimated 12-months PFS and OS was 98%. Grade ≥3 adverse events (AEs) were experienced by 26 patients (31%), whilst 22 serious AEs were observed in 16 patients (19%) (14 infections, 1 febrile neutropenia, 3 dyspnoea, 1 anemia, 1 edema/pleural effusion, 1 renal insufficiency, 1 squamous carcinoma). The most common G3/4 AEs were hematological toxicity (neutropenia [7.1%], anemia [4.5%], thrombocytopenia [2.4%]) and infections (8.5%). The Gr 5 AE consisted of a severe peripheral edema and pleural effusion leading to death. The great majority of SAEs (67%) and G3/4 AEs (66%) were observed during the first 12 cycles of treatment with ibrutinib monotherapy. Conclusions. The preliminary analysis of the GELLC7 trial showed that the addition of consolidation with ofatumumab after 12 cycles of prior treatment with ibrutinib was well tolerated and elicited a deeper response. These results support the potential role of a sequential therapeutic strategy in CLL, where the addition of a consolidation with mAbs in patients with low tumor burden might improve the quality of the response. Finally, more mature results will be further presented at the meeting. Disclosures Abrisqueta: Roche: Consultancy, Honoraria, Other: Travel, Accommodations, expenses, Speakers Bureau; Janssen: Consultancy, Honoraria, Other: Travel, Accommodations, expenses, Speakers Bureau; Celgene: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria, Other: Travel, Accommodations, expenses, Speakers Bureau. González-Barca:Kiowa: Consultancy; Celgene: Consultancy; Takeda: Honoraria; AbbVie: Consultancy, Honoraria; Roche: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Celtrion: Consultancy. Terol:Roche: Consultancy; Janssen: Consultancy, Research Funding; Abbvie: Consultancy; Astra Zeneca: Consultancy; Gilead: Research Funding. Baltasar Tello:GILEAD: Honoraria; JANSSEN: Consultancy, Honoraria; ABBVIE: Honoraria; ROCHE: Honoraria. de la Serna:Roche, AbbVie, Gilead, Janssen, Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Roche, AbbVie, Janssen, Gilead: Speakers Bureau. Ramirez Payer:GILEAD SCIENCES: Research Funding. Cordoba:Janssen: Consultancy, Honoraria, Speakers Bureau; Servier: Consultancy, Honoraria, Speakers Bureau; Kyowa-Kirin: Consultancy, Honoraria, Speakers Bureau; Gilead: Consultancy, Research Funding, Speakers Bureau; Roche: Honoraria, Speakers Bureau; FUNDACION JIMENEZ DIAZ UNIVERSITY HOSPITAL: Employment; Celgene: Consultancy, Honoraria, Speakers Bureau; Pfizer: Consultancy. Bosch:AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Acerta: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; AstraZeneca: Honoraria, Research Funding; Takeda: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; F. Hoffmann-La Roche Ltd/Genentech, Inc.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Kyte: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. OffLabel Disclosure: Ibrutinib followed by Ofatumumab Consolidation