IGHV Gene Replacement: A Potential Mechanism for Establishing Stereotypy in Certain Cases of Chronic Lymphocytic Leukemia

Abstract Immunoglobulin heavy chain variable gene (IGHV) replacement or "VH replacement" (VHR) modifies a rearranged IGHV-D-J sequence by replacing the original IGHV gene with another. This process leaves a detectible "footprint" at the IGHV-D junction of the existing sequence. Roughly 33% of chronic lymphocytic leukemia (CLL) cases exhibit stereotyped B cell receptors (BCRs) often characterized by signature VH CDR3 amino acids. Various mechanisms have been put forth to account for stereotypy in CLL. An overarching hypothesis is that the stereotyped BCRs are antigen driven. Within this concept, a variety of mechanisms could lead to the signatures including somatic mutations and addition/deletion of nucleotides at junctional regions. Here we explore the possibility that VHR provides another mechanism to account for some of the stereotyped rearrangements and some of their signature VH CDR3 amino acid residues in CLL. We examined IG sequences of 26,642 CLL cases and ~16 million healthy controls (HC) to find relic footprints as indicators of VHR. This was done using the VHRFA program developed by Lin Huang et al (PLoS ONE, 2013), as well as our own program which duplicates the VHRFA results but is better able to process large numbers of sequences. The frequency of VHR was similar in CLL and HC (11.6 and 11.9%, respectively). Focusing solely on CLL sequences to define a relationship between VHR and stereotypy, we found highly significant differences in VHR frequencies between stereotyped (n=8,568) and non-stereotyped cases (n=18,074), with stereotyped cases exhibiting VHR at a greatly reduced frequency (7.7% vs. 13.5%, respectively). When comparing VHR frequencies between stereotyped cases and non-stereotyped cases that used the same IGHV, we found that the number of subsets with low VHR exceeded those with elevated VHR ~2:1, accounting for the overall VHR in stereotyped cases being lower than non-stereotyped cases. Further restricting comparisons of stereotyped subsets to non-stereotyped cohorts by matching VH CDR3 length led to similar conclusions. Within stereotyped cases there was a wide distribution of VHR, ranging from 55.6% to 0.1%. Restricting VH CDR3 lengths to "short" (5 - ≤13), "medium" (13.1 - ≤20) and "long" (20.1 - ≤28), the corresponding VHR increased monotonically with length (1.1, 8.2, and 11.9% respectively). Notably, subsets showing elevated VH replacement included better prognosis subsets, #4, 77 and 201 (23.8, 22.1, and 28.6%, respectively). Among low VHR frequency subsets were those associated with worse prognosis, #1, 2, 5, 6, 8, 9 and 10 (VHR frequencies: 0.2, 0.1, 0.9, 2.3, 7.7, 9.0 %, respectively). This was most strikingly exhibited by subsets #1 and #2, both of which comprise patients with poor clinical courses. Each of these sets of sequences displayed virtually no examples of VHR (0.2 and 0.1%, respectively). This might be predicted because these two subsets have relatively short VH CDR3 lengths (subset #1: 13 aa; subset #2: 9 aa), based on the length association mentioned above. Detailed analyses of the presence of footprints and the position of these in the rearranged IGHV-D-J indicated that for some subsets, certain signature VH CDR3 amino acids could be the result of VHR. For example in subset #201, sequence analysis suggests that VHR is responsible for an arginine and for a glutamine in the 5' portion of the VH CDR3. Similarly, VHR may craft the characteristic glutamine on the 5' end of the subset #6 VH CDR3. Thus, our studies indicate that, as a whole, CLL IGHV-D-J sequences use VHR at a frequency comparable to that of normal B cells and significantly less than that of non-stereotyped rearrangements. However, certain stereotyped cases are dramatically enriched for evidence of VHR. Moreover among these cases, the footprints found in the VH CDR3s of stereotyped cases can be shown to directly code for signature amino acids in VH CDR3s. Finally, stereotyped cases with high levels of VHR tend to be those with better clinical courses, whereas those worse outcome stereotyped cases exhibit less evidence for this process. This latter finding is consistent with the concept that VHR is one of the molecular mechanisms used by developing B cells to edit BCRs having high affinity for autoantigens. Since many CLL BCRs are autoreactive, including those found to have high levels of VHR such as subset #4, this implies a fundamental defect in tolerance mechanisms in those normal B cells that eventually became leukemic. Disclosures Agathangelidis: Gilead: Research Funding. Hadzidimitriou:Janssen: Honoraria, Research Funding; Gilead: Research Funding; Abbvie: Research Funding. Ghia:AbbVie, Inc: Honoraria, Research Funding; Acerta: Honoraria, Research Funding; BeiGene: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Gilead: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Sunesis: Honoraria, Research Funding. Stamatopoulos:Gilead: Honoraria, Research Funding; Abbvie: Honoraria, Research Funding; Janssen: Honoraria, Research Funding. Chiorazzi:AR Pharma: Equity Ownership; Janssen, Inc: Consultancy.

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IGHV Gene Replacement in B-Cell Chronic Lymphocytic Leukemia (B-CLL) Occurs at a Frequency Similar to That in Normal B Cells and May Augment Clonal Expansion by Permitting Autogenic/Microbial Clonal Stimulation.

Blood ◽

10.1182/blood.v108.11.2086.2086 ◽

2006 ◽

Vol 108 (11) ◽

pp. 2086-2086

Author(s):

Katerina Hatzi ◽

Sophia Yancopoulos ◽

Emilia Albesiano ◽

Max D. Cooper ◽

Zhixin Zhang ◽

...

Keyword(s):

Amino Acids ◽

B Cells ◽

B Cell ◽

Gene Replacement ◽

Lymphocytic Leukemia ◽

Antigen Receptors ◽

Bacterial Strains ◽

Antigen Specificity ◽

Cell Repertoire ◽

Ighv Gene

Abstract B-CLL cells frequently express antigen receptors with varying degrees of self-reactivity. In this context, it is likely that the precursors of leukemic clones have attempted to reconfigure their receptors to eliminate or reduce affinity for autoantigen. IGHV replacement is a form of receptor reconfiguration that retains the existing IGHD/J rearrangement and swaps the originally rearranged IGHV gene for another upstream IGHV gene, exploiting cryptic heptamer-like sequences in the coding regions of most human IGHV genes. In this study, we explored the extent of IGHV replacement in 583 IGHVDJ rearrangements from patients with B-CLL and compared these findings to normal B cells. B-CLL IGH sequences used IGHV1/2/3/4/5/6/7 subgroup genes in 144/33/255/122/14/9/6 cases, respectively. IGHV3 and IGHV4 genes were used predominantly in mutated (M) sequences (136/255 and 75/122 cases respectively); in contrast, 83% (120/144) of IGHV1 genes were used in unmutated (UM) sequences. IGHV replacement in B-CLL was examined using a search method, also applied in the normal repertoire, looking for the presence of pentameric footprints, which are short stretches of 3′ nucleotides from the original IGHV that remain in the IGHVD joint after each round of IGHV replacement. This method uses a set of 33 pentamers deriving from the 3′ends of human IGHV genes carrying cryptic heptamers to identify potential IGHV replacement products. After alignment with sequences in the IGHVD junctions in B-CLL, only exact matches were accepted. At least one pentameric footprint consistent with IGHV replacement was detected in the IGHVD junction of 34 (19 M/15 UM) of 583 B-CLL cases (5.8%). Twenty-seven pentamers were detected in this cohort of sequences. B-CLL rearrangements with evidence of IGHV replacement footprints had longer HCDR3 regions (median,19 amino acids) compared to the series as a whole (median,17 amino acids). The most frequent IGHV genes that probably replaced a previously rearranged IGHV gene were IGHV1-69 (6 cases/5UM), IGHV3-23 (6 cases/1UM), IGHV3-7 (4 cases/1UM), IGHV3-33 (3 cases/2UM). In 30/34 cases (88%), the amino acids encoded by these footprint sequences in the HCDR3 where positively or negatively charged, a finding associated with autoreactivity. These residues likely contribute to antigen specificity and affinity and can be important for B-CLL cells, which are antigen-experienced, activated cells. In conclusion, IGHV replacement in CLL appears to occur at a frequency (5.8%), which is not inherently different from that reported in the normal B-cell repertoire (5%). Although IGHV replacement is ostensibly initiated to modify autoreactivity, its products still often resemble autoantibodies (increased HCDR3 length, aminoacid composition and charge). However since some of these HCDR3 properties also exist in anti-microbial mAbs, their retention after secondary rearrangements may be a protective as well as a potentially harmful mechanism. Such a selection for microbial reactivity supports the notion that B-CLL derives from cells expressing mAb with shared reactivity between autoantigens and microbes. In support of this possibility is the finding that a UM-IGHV1-69 recombinant mAb with evidence of IGHV replacement reacted against 5 different bacterial strains.

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Dysregulation of TNFα-Induced Necroptotic Signaling In Chronic Lymphocytic Leukemia: Suppression of CYLD Gene by LEF1

Blood ◽

10.1182/blood.v116.21.377.377 ◽

2010 ◽

Vol 116 (21) ◽

pp. 377-377 ◽

Cited By ~ 2

Author(s):

Peng Liu ◽

Bei Xu ◽

Jianyong Li

Keyword(s):

Cell Death ◽

B Cells ◽

Chronic Lymphocytic Leukemia ◽

Molecular Mechanisms ◽

Conflicts Of Interest ◽

Death Receptors ◽

Lymphocytic Leukemia ◽

Ros Production ◽

A Genome ◽

Stimulation Of

Abstract Abstract 377 Impaired cell death program has been noted as one of the hallmarks of Chronic lymphocytic leukemia (CLL) and contributes to its accumulation of malignant monoclonal B cells as well as to chemotherapy resistance. A cell can die through apoptosis or necrosis pathway. While apoptosis is known as a regulated cellular program, necrosis is known as an accidental event caused by overwhelming stress. However, accumulating evidence suggests that necrosis can also be executed by regulated mechanisms, especially in apoptotic-deficient conditions. Recently, the term necroptosis has been used to designate one particular form of programmed necrosis induced by stimulating death receptors with agonists such as TNFα, FasL, and TRAIL. Apoptosis suppression by caspase inhibitors such as zVAD may switch apoptotic response to necroptosis or enhance necroptosis. In contrast to well-characterized apoptotic pathway, the detailed molecular mechanisms underlying necroptosis are still not fully understood. A genome wide siRNA screen revealed two members of the receptor interacting protein (RIP) kinase family, RIP1 and RIP3P, to be essential for necroptosis. Upon stimulation of death receptors, RIP3 is recruited to RIP1 to form a necroptosis-inducing complex which is essential for cell death execution. The deubiquitinase cylindromatosis (CYLD) is recruited to TNFα receptor upon its activation and directly regulates RIP1 ubiquitination. In addition, by activating key enzymes of metabolic pathways, RIP3 regulates TNFα-inducing mitochondrial reactive oxygen species (ROS) production, which partly accounts for its ability to potentiate necroptosis. Until now, much less is known about the significance of necroptosis in malignant disease. Here we demonstrate that primary CLL cells failed to undergo necroptosis upon stimulation of TNFα combined with pan-caspase inhibitor zVAD. Upon TNFα+zVAD stimulation, normal CD19+ B cells increased ROS production > 8 fold, while same treatment only resulted in ∼ 2 fold induction in ROS generation in CLL samples. Two core components of necroptotic machine, RIP3 and CYLD, are markedly down-regulated in CLL compared with normal B cells, at both protein and transcription levels. Moreover, we identified LEF1, a downstream effector of Wnt/β-catenin pathway, as a transcription repressor of CYLD in CLL. LEF1 is highly expressed in CLL cells, whereas normal B cells have very low levels of LEF1 expression. Attenuation of LEF1 expression through RNAi technology resulted in a dramatic increase in CYLD levels in CLL cells, as determined by western blot and real time RT-PCR analysis. Dual-luciferase assays showed that forced expression of LEF1 markedly decreased CYLD promoter activity compared with controls. Mutation of LEF1 responsive elements (LERs) on CYLD promoter significantly abolished transcriptional repression of CYLD by LEF1. Chromatin immunoprecipitation assays showed that LEF1 is recruited to LER region within the CYLD promoter in CLL cells. Additionally, Knocking down LEF1 sensitizes CLL cells to TNFα-induced necroptosis. The present investigation provides the first evidence that CLL cells have defects not only in apoptotic program but also in necroptotic signaling. Targeting the key regulators of necroptotic machine such as LEF1 to restore this pathway may represent a novel approach for CLL treatment. Disclosures: No relevant conflicts of interest to declare.

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Increased Activity of Aldehyde Dehydrogenase, a Stem/Progenitor Cell Marker, in Chronic Lymphocytic Leukemia,

Blood ◽

10.1182/blood.v118.21.3867.3867 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3867-3867

Author(s):

Raymond P. Wu ◽

Christina C.N. Wu ◽

Tomoko Hayashi ◽

Laura Z. Rassenti ◽

Thomas J. Kipps ◽

...

Keyword(s):

B Cells ◽

Chronic Lymphocytic Leukemia ◽

Board Of Directors ◽

Research Funding ◽

Progenitor Cell ◽

Lymphocytic Leukemia ◽

Cell Marker ◽

Aldh Activity ◽

Advisory Committees ◽

Aldefluor Assay

Abstract Abstract 3867 Introduction: Despite their mature appearance, the B cells from chronic lymphocytic leukemia (CLL) possess immature characteristics both functionally and biochemically. CLL B cells display known biochemical markers characteristic of cells early in the blood lineage, including ROR1, Wnt16, and LEF1. In addition, CLL B cells have higher levels of Reactive Oxygen Species (ROS) and of the oxidant-induced transcription factor Nrf2 [NFE2L2], compared to normal peripheral blood mononuclear cells (PBMC). Intracellular ROS status has been suggested to be a marker of cancer stem/progenitor cells possibly due to their high expression of oncogenes. Downstream targets of Nrf2 include the Aldehyde dehydrogenase [ALDH] enzymes, which are believed to play a crucial role in stem cell biology because they protect the cells against oxidative stress caused by accumulation of aldehydes. Here, we use ALDH activity to visualize populations of CLL B cells that may have stem/progenitor properties. Materials and Methods: Isolated PBMC from normal donors and CLL patients with aggressive and indolent disease were stained for ALDH activity with an Aldefluor assay kit (StemCell Technologies). The ALDH inhibitor, diethylaminobenzaldehyde (DEAB), was used to confirm that the fluorescent activity was due to ALDH activity. At the end of the Aldefluor assay, the cells were stained for cell surface markers, CD19, CD5, CD38 and CD34. 50,000 total events were collected for FACS analysis. Normalized Mean Fluorescence Intensity (MFI) values were calculated by dividing each MFI value to average MFI value of normal CD19+ cells for each experiment. Data analyses were performed by FlowJo software and Prizm. P-values were calculated by One-Way ANOVA analysis with Post-Bonferroni's multiple comparison test. Results: We examine the level of ALDH expression and activity in CD19+ cells of healthy donors (n = 9), CLL samples that expressed unmutated IgVH and that were ZAP-70 positive (defined as “aggressive”, n = 14) or samples that expressed mutated IgVH and were ZAP-70 negative (defined as “indolent”, n=12). CLL B cells from patients with aggressive disease had significantly higher ALDH activities compared to normal B cells (p < 0.001) and indolent CLL B cells (p < 0.05) (Figure1). Indolent CLL B cells also have higher level of ALDH activities compared to normal B cells (p < 0.01) (Figure1). Treatment with the ALDH inhibitor, DEAB, suppressed the increased fluorescence observed in CLL B cells. In addition, ALDH high CLL B cells are CD34 negative. These data show that CLL B cells express a marker known to be associated with stem/progenitor cells, but these populations are different from CD34 positive hematopoietic stem cells. In addition, our data show that a stem/progenitor cell marker is associated with the pathogenesis of CLL. Disclosures: Kipps: Igenica: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Research Funding; Abbot Industries: Research Funding; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees; Genentech: Research Funding; GSK: Research Funding; Gilead Sciences: Consultancy, Research Funding; Amgen: Research Funding.

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Survivin is expressed on CD40 stimulation and interfaces proliferation and apoptosis in B-cell chronic lymphocytic leukemia

Blood ◽

10.1182/blood.v97.9.2777 ◽

2001 ◽

Vol 97 (9) ◽

pp. 2777-2783 ◽

Cited By ~ 243

Author(s):

Luisa Granziero ◽

Paolo Ghia ◽

Paola Circosta ◽

Daniela Gottardi ◽

Giuliana Strola ◽

...

Keyword(s):

B Cells ◽

Chronic Lymphocytic Leukemia ◽

B Cell ◽

Molecular Mechanisms ◽

Mononuclear Cells ◽

Survivin Expression ◽

Lymphocytic Leukemia ◽

Cd40 Stimulation ◽

Cell Chronic Lymphocytic Leukemia

Abstract In B-cell chronic lymphocytic leukemia (B-CLL), defective apoptosis causes the accumulation of mature CD5+ B cells in lymphoid organs, bone marrow (BM), and peripheral blood (PB). These cells are the progeny of a proliferating pool that feeds the accumulating compartment. The authors sought to determine which molecular mechanisms govern the proliferating pool, how they relate to apoptosis, and what the role is of the microenvironment. To begin to resolve these problems, the expression and modulation of the family of inhibitor of apoptosis proteins (IAPs) were investigated, with consideration given to the possibility that physiological stimuli, such as CD40 ligand (CD40L), available to B cells in the microenvironment, might modulate IAP expression. The in vitro data on mononuclear cells from PB or BM of 30 patients demonstrate that B-CLL cells on CD40 stimulation express Survivin and that Survivin is the only IAP whose expression is induced by CD40L. Through immunohistochemistry, in vivo Survivin expression in lymph node (LN) and BM biopsies was evaluated. In reactive LN, Survivin was detected only in highly proliferating germinal center cells. In LN from patients with B-CLL, Survivin was detected only in pseudofollicles. Pseudofollicle Survivin+ cells were actively proliferating and, in contrast to Survivin+ B cells found in normal GC, were Bcl-2+. In B-CLL BM biopsies, CD5+, Survivin+ cells were observed in clusters interspersed with T cells. These findings establish that Survivin controls the B-CLL proliferative pool interfacing apoptosis and that its expression may be modulated by microenvironmental stimuli.

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Intraclonal Diversification Occurs in Chronic Lymphocytic Leukemia Expressing B Cell Receptors Belonging to the IGHV4 Gene Family

Blood ◽

10.1182/blood-2018-99-112423 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 944-944

Author(s):

Filippo Vit ◽

Francesca Maria Rossi ◽

Tiziana D'Agaro ◽

Tamara Bittolo ◽

Antonella Zucchetto ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Research Funding ◽

Excision Repair ◽

Lymphocytic Leukemia ◽

Mrna Levels ◽

Direct Role ◽

Region Gene ◽

Mutational Status ◽

Ighv Gene

Abstract Background. The pivotal role of the Immunoglobulin (Ig) receptor and antigenic stimulation have been proven to be landmarks for the understanding of the ontogeny and evolution of chronic lymphocytic leukemia (CLL). In addition, the mutational status of the Immunoglobulin Heavy-Chain Variable region gene (IGHV) was confirmed to be a reliable prognostic factor, supporting an antigen-driven model of CLL development. To clarify aspects regarding an antigenic involvement in CLL evolution, studies focusing on intraclonal diversification (ID) of Ig genes have provided relevant information, although mainly conducted in a pre-Next Generation Sequencing (NGS) era. Aim. To apply a NGS approach to investigate ID in CLL. Methods. IGHV genes from 530 CLL patients with Royal Masden Hospital score 4-5 (Fig. 1A) was sequenced using NGS (Lymphotrack). The bio-informatic pipeline was based on the pRESTO/ChangeO packages. Specific pathological clones were selected based on the presence of same IGHV, junction genes and with similar HCDR3 sequence according to Hamming's distance. Through the R-Alakazam package, we generated rarefaction curves to evaluate the clonal diversity inside the pathological clone (Fig. 1B). Focusing on the Simpson index (represented by the Hill number of order q=2), which gives more weight to larger clones minimizing the smaller ones (Fig. 1B), we selected a Diversity Score (DS) of 4 for the definition of cases without ID (clonal; DS <4) and cases with ID (intraclonal; DS ≥4) (Fig. 1B). Results. Using the reported threshold we identified 469 (88.5%) clonal cases, expressing a single clone (Fig. 1C), and 61 (11.5%) cases with ID (median DS 9.2, range 4.4-66.0) characterized by the presence of two or multiple pathological clones expressing the same IGHV gene and HCDR3 (Fig. 1C). Notably, cases with ID expressed both a mutated (M) (39/61, 63.9%) and an unmutated (UM) (22/61, 36.1%; p=0.066) IGHV gene configuration (Fig. 1C). Of note, we observed a significant skewing toward the usage of VH4-family genes when comparing cases with ID (38/61, 62.3%) vs. cases without ID (78/469, 16.6%; p<0.0001, Fig 1D). Moreover, the IGHV4-39 and IGHV4-34 genes were the most used genes in the context of cases with ID (Fig. 1E), although none of them belonging to known stereotyped subsets. By focusing on VH4-family only cases, we observed that cases with ID and UM IGHV genes displayed higher mutation frequencies in WA/TW motifs, a mutational signature which suggests an involvement of both Activation-Induced (Cytidine) Deaminase (AID) and error-prone polymerase eta (Fig. 1F), a pattern not observed in its counterpart with UM IGHV genes but without ID (Fig. 1F). Conversely, in cases with ID and M IGHV genes, mutations preferentially clustered in AID hotspots (WRC/GYW motifs), suggesting a direct role of AID and the Base Excision Repair machinery in the mutational overload (Fig. 1F). Consistently, M IGHV cases with ID expressed significantly higher AID mRNA levels than M IGHV cases without ID (p=0.0024; Fig. 1G). These expression levels were overall comparable with those found in UM IGHV cases, irrespective to the evidence of ID (Fig. 1G), which however were not associated with an increased number of mutations in AID-specific hotspots (Fig. 1F). Conclusions. By taking advantage of a new method for ID assessment in CLL, we demonstrated that ID prevalently affects VH4-family cases which display different mutational patterns dependent to the IGHV gene status. This data are in keeping with previous reports indicating the IGHV4 genes as particularly prone to generate immunoglobulin subjected to continuous/persistent stimulation by external/auto-antigens, hence particularly prone to generate features of ID. Further experiments in selected cases with ID through a non-random barcode strategy are needed. Disclosures Zaja: Sandoz: Honoraria; Celgene: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Amgen: Honoraria; Janssen: Honoraria; Takeda: Honoraria; Abbvie: Honoraria.

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Bone Marrow Hematopoietic Dysfunction in Untreated Chronic Lymphocytic Leukemia Is Partially Mediated By Exposure to Constituents of the Leukemic Microenvironment

Blood ◽

10.1182/blood-2018-99-118916 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 3132-3132

Author(s):

Bryce Manso ◽

Kimberly Gwin ◽

Charla R Secreto ◽

Henan Zhang ◽

Wei Ding ◽

...

Keyword(s):

Bone Marrow ◽

B Cells ◽

Chronic Lymphocytic Leukemia ◽

Board Of Directors ◽

Research Funding ◽

Lymphocytic Leukemia ◽

Healthy Controls ◽

Advisory Committees ◽

The Impact

Abstract Peripheral immune dysfunction in B-Chronic Lymphocytic Leukemia (CLL) is well-studied and likely relates to the incidence of serious recurrent infections and second malignancies that plague CLL patients. However, the current paradigms of known immune abnormalities are not able to consistently explain these complications and it is not easy to correct CLL patient immune status. Here, we expand on our preliminary reports that demonstrate bone marrow (BM) hematopoietic dysfunction in early and late stage untreated CLL patients. We found reduced short-term functional capacity of hematopoietic progenitors in BM using colony forming unit assays (Figure 1A-C) and flow cytometry revealed significant reductions in frequencies of hematopoietic stem and progenitor cell (HSPC) populations (exemplified by Lin-CD34+ HSPCs, Figure 1D). We further report that protein levels of the transcriptional regulators HIF-1α, GATA-1, PU.1, and GATA-2 are overexpressed in distinct HSPC subsets from CLL patient BM, providing molecular insight into the basis of HSPC dysfunction. Interestingly, sustained myelopoiesis, evaluated by limiting dilution analysis in long-term culture-initiating cell (LTC-IC) assays maintained for five weeks, revealed no difference between healthy controls and CLL patients. These new data indicate that when HSPCs are removed from the leukemic microenvironment for ample in vitro culture time, they recover the ability to sustain myelopoiesis. To further assess the impact of the CLL microenvironment on HSPC biology, isolated HSPCs (CD34+ BM cells) from healthy controls were exposed in vitro to known leukemic microenvironment constituents. Exposure to TNFα, a cytokine constitutively produced by CLL B cells, resulted in rapid increases in PU.1 and GATA-2 proteins (Figure 2A-D). Similarly, addition of TNFα to the LTC-IC assay resulted in a striking ablation of myelopoiesis, even at the highest input cell concentration. Further, overexpression of PU.1 and GATA-2 were observed in HSPCs following co-culture with CLL B cells, a result that was not recapitulated when cells were exposed to IL-10, another cytokine constitutively produced by CLL B cells. These findings indicate specific components of the leukemic microenvironment are involved in HSPC modulation. Together, these findings expand on our previous observations of BM hematopoietic dysfunction in untreated CLL patients and offer new molecular insights into the contribution of the leukemic microenvironment on immunodeficiency in CLL. Disclosures Ding: Merck: Research Funding. Parikh:Pharmacyclics: Honoraria, Research Funding; MorphoSys: Research Funding; Janssen: Research Funding; Abbvie: Honoraria, Research Funding; Gilead: Honoraria; AstraZeneca: Honoraria, Research Funding. Kay:Morpho-sys: Membership on an entity's Board of Directors or advisory committees; Agios Pharm: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Acerta: Research Funding; Infinity Pharm: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Tolero Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Cytomx Therapeutics: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding.

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Calcium Mobilization in Unfavorable-Prognosis Chronic Lymphocytic Leukemia Patients Mediates Focal Adhesion Kinase (FAK) Cleavage, Thereby Its Activation

Blood ◽

10.1182/blood-2018-99-114492 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 5537-5537

Author(s):

Federica Frezzato ◽

Veronica Martini ◽

Filippo Severin ◽

Flavia Raggi ◽

Marco Piccoli ◽

...

Keyword(s):

Flow Cytometry ◽

B Cells ◽

Chronic Lymphocytic Leukemia ◽

Focal Adhesion Kinase ◽

Focal Adhesion ◽

Research Funding ◽

Lymphocytic Leukemia ◽

Full Length ◽

Dependent Manner ◽

Significant Difference

Abstract INTRODUCTION Chronic Lymphocytic Leukemia (CLL) is the most common leukemia in the western world and is characterized by the accumulation of monoclonal B cells, due to both increased proliferation and apoptosis resistance. Although in the last years with the introduction of new kinase inhibitors blocking the pathways mediated by B-cell receptor (BCR) signaling we got an astonishing progress in the comprehension and treatment of this disease, CLL is still an incurable disease and many characteristics of its pathogenesis still remain unclear. Signaling events downstream the BCR engagement are central for the progression of CLL. Focal Adhesion Kinase (FAK), one of the primary enzyme involved in the engagement of integrins and assembly of focal adhesions, plays a major role in cellular adhesion and metastasis of various cancers, being regulated by Calcium (Ca2+) flux and by Src-kinases (e.g. Lyn) through a Calpain-dependent manner following BCR triggering. FAK has been demonstrated to be over-expressed in many human cancers but a down-modulation of its expression has also been reported. Studies concerning FAK expression in CLL are lacking in the literature. However, since an interaction of FAK with molecules implicated in BCR signal transduction, such as the Src-kinase Lyn, has been demonstrated we hypothesize that this kinase could have a key role in CLL pathogenesis. METHODS FAK expression was analyzed in B-lymphocytes from 107 CLL patients and 10 healthy subjects by Western blotting (WB) and the obtained expression data were correlated with the clinical features of the patients. In 25 out of 107 patients studied, surface IgM and IgD expression has been evaluated by flow cytometry (FC). For Ca2+ mobilization assessment, 1x107 cells were incubated with 4μM Fluo-4-AM at 37°C for 30min and then analyzed by FC; after 30s of baseline acquisition, α-IgM F(ab')2 and α-IgD F(ab')2 (10μg/ml) were added and fluorescence intensity was recorded for 5min. Ionomycin was added as positive control. Phosphorylation at Tyr397 was assessed with a specific antibody. Leukemic B cells from patients were treated in vitro with 5μM Defactinib (FAK inhibitor) and the apoptosis induction was evaluated by Annexin V/Propidium Iodide flow cytometry test and by the presence of cleaved PARP by WB. RESULTS By WB analyses we demonstrated a slightly significant difference in FAK expression between patients and controls (p<0.05), the protein being particularly down-regulated in unmutated IGHV and del17p/del11q/12+ CLL patients. We observed that FAK down-modulation was limited to its whole form detected in WB at 125kDa, while bands related to FAK cleavage (92/94 and 84kDa) were detected also in those patients lacking full length-FAK. Cleaved-FAK is due to Calpain protease activity, when stimulated by the bond with Ca2+ ions. We then compared FAK expression with the capability of the cell to mobilize Ca2+ from intracellular stores, observing that patients with this capability had less amount of full length-FAK, which translated into a higher presence of cleaved/activated form of FAK. The cleavage bands infact were found phosphorylated at activatory Tyr397. Of note, only IGHV-unmutated patients showed these features. Lastly, we studied the effect of Defactinib, a specific FAK inhibitor, in CLL cells; this molecule was able to induce apoptosis in leukemic cell in a caspase-dependent way, as assessed by the presence of the cleaved PARP. CONCLUSIONS We herein propose that full length-FAK down-modulation could be considered as a new marker of unfavorable prognosis. In our model, poor prognosis CLL patients (particularly IGHV unmutated ones) presenting Ca2+ mobilization, are more prone to activate Calpain, which in turn activates FAK. Together with data from the literature, our results suggest that CLL cells missing the full length-FAK, not only are unaffected by the lack of it, but they rather present a cleaved/activated form of FAK that could favor cell migration and metastatic invasion. Moreover, since Defactinib can induce apoptosis in CLL cells, should these data be confirmed by in vivo studies, this FAK inhibitor could represent a new therapeutic approach for CLL. Disclosures Trentin: Gilead: Research Funding; Abbvie: Honoraria; Roche: Membership on an entity's Board of Directors or advisory committees; Janssen: Research Funding.

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Indoleamine 2,3-Dioxygenase Mediates Survival of Chronic Lymphocytic Leukemia B Cells through Aryl Hydrocarbon Receptor By Inducing Mcl1

Blood ◽

10.1182/blood-2020-142345 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 19-19

Author(s):

Claudio Giacinto Atene ◽

Rossana Maffei ◽

Stefania Fiorcari ◽

Silvia Martinelli ◽

Patrizia Zucchini ◽

...

Keyword(s):

T Cells ◽

B Cells ◽

Chronic Lymphocytic Leukemia ◽

Aryl Hydrocarbon Receptor ◽

Research Funding ◽

Lymphocytic Leukemia ◽

Leukemic Cells ◽

Strong Increase ◽

Indoleamine 2,3 Dioxygenase ◽

Aryl Hydrocarbon

Introduction: Chronic lymphocytic leukemia (CLL) is a dynamic disease in which monoclonal B cells proliferate within the pseudo-follicular centers in lymphoid organs and then they accumulate due to an intrinsic defect of apoptosis. Leukemic cells are considered as "addicted to the host" since extrinsic signals from the microenvironment strongly influence the establishment of a progressive immunosuppression for malignant cell growth and survival. The cytoplasmic enzyme indoleamine 2,3-dioxygenase (IDO) mediates the conversion of the essential amino acid tryptophan (Trp) into metabolic byproducts such as kynurenine (Kyn). Kyn and other secondary metabolites are endogenous activators of the aryl hydrocarbon receptor (AHR), a ligand-controlled transcription factor that mediates cellular responses to toxins or endogenous ligands. The IDO-Kyn-AHR axis plays important roles in carcinogenesis and cancer progression. The mechanisms that promote inflammation around tumor tissues and determine immune tolerance consist in Trp depletion, which induces T cell apoptosis, and in Kyn-mediated AHR activation that inhibits effector T cells and promotes regulatory T cells differentiation. IDO protein is expressed in human hematologic malignancies and its level is correlated with a poor prognosis and chemoresistance. The IDO activity, measured as the Kyn/Trp ratio, was reported to be increased in CLL cases comparing to normal controls. Aim: We wondered to characterize the expression of IDO and AHR in CLL patients and to dissect the biological function of the IDO-Kyn-AHR axis. Methods: Gene transcription and protein expression were evaluated by real time PCR and western blot. Enzymatic activity was assessed through ELISA. Survival was measured with PI/annexin V assay. Overexpression and silencing of target genes was obtained by nucleofection. Results: Firstly, we observed that CLL cells expressed both IDO and AHR at variable levels. Moreover, we found that several microenvironmental signals such as IFNγ, LPS, anti-IgM, CpG oligo DNA, CD40L and TNFα were able to up-regulate IDO and AHR mRNA and protein. To characterize the pathways able to mediate IDO expression, we stimulated CLL cells with IFNγ and CD40L. Using ruxolitinib, an inhibitor of JAK-STAT pathway, we found that IFNγ induced IDO through STAT1 signaling. Again, CD40L stimulation determined IDO overexpression through the non-canonical NF-kB pathway, as assessed by treating cells with NF-κB inducing kinase inhibitor, NIK SMI1. We also confirmed that IFNγ-treated CLL cells were able to produce a functional IDO enzyme by measuring Kyn production and Trp consumption by ELISA. The strong increase in the Kyn/Trp ratio induced by IFNγ was significantly reduced by ruxolitinib treatment. To verify if Kyn produced by CLL cells could act through an autocrine loop on AHR, leukemic cells were treated with Kyn. We observed that Kyn mediated AHR translocation from the cytoplasm to the nuclei, inducing its activation as assessed by up-regulation of CYP1A1, a known AHR target gene. Of interest, we found that Kyn treatment improved CLL cells survival. Analyzing the anti-apoptotic proteins of the Bcl2 family after Kyn treatment, we found the induction of Mcl1, that was affected by adding CH-223191, an antagonist of AHR. Moreover, we transfected CLL cells with an IDO vector. The up-regulation of IDO increased CLL cells survival through the induction of Mcl1. Accordingly, when CLL cells were silenced for AHR, we observed a reduction of their survival. Conclusion: Our data demonstrate the constitutive expression of IDO and AHR in CLL cells. Furthermore, the tumor microenvironment promotes the induction of IDO and AHR through a complex signaling crosstalk with leukemic cells. Our findings underline that IDO-Kyn-AHR axis is active in CLL cells and promotes Mcl1 expression, sustaining the survival of CLL cells. Disclosures Luppi: Gilead Sci: Consultancy, Speakers Bureau; MSD: Consultancy; Sanofi: Consultancy; Abbvie: Consultancy; Daiichi-Sankyo: Consultancy; Novartis: Consultancy, Speakers Bureau. Marasca:Gilead Sci: Honoraria, Research Funding; Roche: Consultancy, Honoraria; Shire: Consultancy, Honoraria; Janssen: Honoraria, Research Funding; Abbvie: Consultancy, Honoraria.

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Lenalidomide-Dependent Activation of the Phosphatidylinositol 3-kinase-δ Pathway Is Antagonized by CAL-101 In Chronic Lymphocytic Leukemia

Blood ◽

10.1182/blood.v116.21.1821.1821 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1821-1821

Author(s):

Sarah E.M. Herman ◽

Rosa Lapalombella ◽

Jeffrey A. Jones ◽

Leslie Andritsos ◽

Amber L. Gordon ◽

...

Keyword(s):

B Cells ◽

Chronic Lymphocytic Leukemia ◽

Research Funding ◽

Cell Activation ◽

Specific Inhibitor ◽

Lymphocytic Leukemia ◽

Clinical Activity ◽

Cytokine Release ◽

Treatment Naïve ◽

Previously Treated

Abstract Abstract 1821 Lenalidomide is an oral immune-modulating agent that has been shown to have clinical activity in patients with treatment-naive and previously treated chronic lymphocytic leukemia (CLL). In CLL, a disease-specific phenomenon of drug-induced tumor flare is often observed that results in lymph node enlargement, rash, and cytokine release. We and others have attributed both lenalidomide-induced tumor flare and cytokine release in part to CLL cell activation, with concomitant increase in surface co-stimulatory molecules including CD154. The potential consequences of such activation by lenalidomide in CLL are multiple. In symptomatic, previously untreated CLL, activation of tumor cells by lenalidomide likely contributes to reversal of hypogammaglobulinemia in a subset of patients. Additionally, activation of CLL cells increases their capacity for antigen presentation, potentially facilitating a clinically beneficial development of tumor-specific antibodies toward antigens such as ROR1. In patients with previously treated CLL, lenalidomide therapy does not reverse hypogammaglobulinemia. However, treatment has been documented to increase serum b-FGF and VEGF levels, which correlates with lack of response. Previous work demonstrates that CLL cells predominately utilize the PI3K p110δ isoform for activation following CD154 signaling. Given our prior findings of prominent lenalidomide induction of CD40-CD154 signaling in vitro and in vivo, we focused initially on molecular interrogation of isoforms responsible for this in CLL cells. Utilizing primary CLL cells, we demonstrated that inhibition of PI3K-δ signaling by CAL-101, a clinically relevant PI3K-δ isoform-specific inhibitor, abrogated lenalidomide-induced activation of CLL cells by directly reducing PI3K enzymatic activity and also reducing phosphorylation of the downstream PI3K target AKT. Parallel studies with siRNA targeted to the p110δ isoform of PI3K demonstrated antagonism of lenalidomide-induced AKT phosphorylation. Furthermore, we found that inhibition of PI3K-δ by CAL-101 at therapeutically relevant concentrations (1 μM) prevented up-regulation of CD40, CD154, and CD86 by lenalidomide and also antagonized production of IgM by normal B-cells co-cultured with CLL cells. Collectively, these data demonstrate the importance of PI3K-δ signaling in modulating the pharmacological effects of lenalidomide in CLL cell activation including up-regulation of CD40, CD154, CD86 and active CLL cell co-stimulation of normal B-cells. Our findings suggest that clinical evaluation of combination strategies of lenalidomide and CAL-101 in treatment-naive patients with CLL should be performed with careful pharmacodynamic monitoring of immune modulation and signaling to best preserve the clinical benefits of both drugs. This work is supported by the Leukemia and Lymphoma Society, D. Warren Brown Foundation, and The OSU Leukemia SPORE grant funded by the NCI. CAL-101 was provided by Calistoga Pharmaceuticals, Inc. Disclosures: Jones: Glaxo Smith-Kline: Consultancy; Abbott: Research Funding. Lannutti:Calistoga Pharmaceutical Inc.: Employment. Byrd:Calistoga Pharmaceutical Inc.: Equity Ownership. Johnson:Calistoga Pharmaceutical Inc.: Research Funding.

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Efficient Gene Delivery to Chronic Lymphocytic Leukemia (CLL) Cells with Microbubbles Bearing ROR1 Antibody

Blood ◽

10.1182/blood.v118.21.2857.2857 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2857-2857

Author(s):

Suping Zhang ◽

Wenjin Cui ◽

Robert Mattrey ◽

Thomas J. Kipps

Keyword(s):

B Cells ◽

Chronic Lymphocytic Leukemia ◽

Duty Cycle ◽

Research Funding ◽

Ex Vivo ◽

Lymphocytic Leukemia ◽

Ultrasound Treatment ◽

Targeted Microbubbles ◽

Receive Treatment

Abstract Abstract 2857 Patients of chronic lymphocytic leukemia (CLL) typically develop progressive immune deficiency, which impairs their response to vaccines. Prior studies showed that infusions of autologous CLL cells transduced ex vivo with adenovirus encoding CD154 could elicit anti-leukemia immune responses. However, the need for high amounts of high-titer adenovirus complicates this approach and handicaps clinical development. A recently developed technology, known as microbubbles, could be activated using safe ultrasound (U/S) energy, potentially providing a new tool with which to affect gene delivery. In this study, we examined whether microbubble-technology could effectively transfect CLL B cells ex vivo or in vivo. To target CLL cells with microbubbles, we used newly generated mAb specific for ROR1, a surface antigen expressed on CLL B cells, but not on normal B cells or most other adult tissues. Anti-ROR1 mAb was incorporated into the lipid shell of microbubble, which was composed with DSPC and DSPE-PEG2000-Maleimide via maleimide chemistry. We examined whether targeting the microbubbles with anti-ROR1 mAb improved our transfection efficiency for ROR1+ CLL cells. For this we generated microbubbles tagged with anti-ROR1 mAb or control Ig and incubated these with fresh whole blood from patients with CLL. We found that targeted bubbles specifically bound to CD19+CD5+ CLL cells, but not to other cells, including RBCs. Next we examined whether anti-ROR1-tagged microbubbles or non-targeted microbubbles could transfect CLL cells with an expression plasmid (pGFP) encoding green-fluorescence protein (GFP). Isolated CLL cells were incubated with pGFP-laden microbubbles for 1 hour at 37° C. Following this the preparations were exposed to ultrasound at 2w/cm2 and 50% duty cycle or left untreated. After 48 hours culture, the cells were collected and stained with propidium iodide (PI) and examined via flow cytometry. Using these conditions, CLL cells in all treatment groups retained high viability (>80%). CLL cells incubated with anti-ROR1 mAb-tagged microbubbles expressed high levels of GFP provided they had also been exposed to ultrasound; CLL cells treated with anti-ROR1-mAb-tagged microbubbles that did not receive ultrasound treatment remained negative for expression of GFP. Similarly, CLL cells treated with non-targeted pGFP-laden microbubbles did not express GFP regardless of whether they also received ultrasound treatment. We also examined whether anti-ROR1-mAb-tagged microbubbles could transfect CLL cells in vivo. For this 107 human CLL B cells were injected into the peritoneum of each RAG-2−/−/ γc−/− mouse. Five minutes later, anti-ROR1-mAb-tagged microbubbles or non-targeted microbubbles laden with expression vectors encoding GFP and luciferase were injected into the peritoneum of each animal. Ten minutes later groups of animals did or did not receive treatment with ultrasound at 2w/cm2 and 50% duty cycle for 1 min. Forty-eight hours later mice were examined for reporter gene expression via whole body imaging. The group of animals that received anti-ROR1-mAb-tagged microbubbles and ultrasound treatment all had high-level expression of luciferase. Cells were recovered from the mice via peritoneal lavage. Immunofluorescence studies confirmed that only ROR1+CD5+ cells expressed GFP. In contrast mice injected with anti-ROR1-mAb-tagged microbubbles but did not receive treatment with ultrasound had no detectable luciferase activity. Groups of mice that were treated with non-targeted microbubbles also had negligible leuciferase activity regardless of whether they received ultrasound treatment. This study reveals that anti-ROR1-mAb tagged microbubbles that are activated by extracorporeal ultrasound treatment can be effective vehicles for specific delivery of genes into CLL cells ex vivo and in vivo. Disclosures: Kipps: Igenica: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Research Funding; Abbott Industries: Research Funding; Genentech: Research Funding; GSK: Research Funding; Gilead Sciences: Consultancy, Research Funding; Amgen: Research Funding.

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