Murine Model Of Chronic Lymphocytic Leukemia Research Articles

Chronic Lymphocytic Leukemia Cells with Mutated Nfkbie Are Positively Selected By Microenvironmental Signals and Display Reduced Sensitivity to Ibrutinib Treatment

Inactivating mutations in NF-kB pathway genes, such as the NF-kB inhibitor NFKBIE, are among the more frequent genetic lesions in chronic lymphocytic leukemia (CLL). However, the role of these genetic lesions in CLL pathogenesis and treatment resistance is still largely unknown and requires further study in in vivo models of the disease. To this end, we generated transplantable murine leukemias with inactivating NFKBIE mutations and investigated their impact on leukemia growth and response to ibrutinib (IBR) treatment. The NFKBIE mutations were introduced by CRISPR/Cas9 editing in two recently established autoreactive leukemia lines derived from the Eμ-TCL1 murine CLL model. These cell lines proliferate spontaneously in vitro in a BCR-dependent manner, but also respond with increased proliferation to certain microenvironmental signals, such as those generated by Toll-like receptor (TLR) stimulation (Chakraborty S et al, Blood 2021).To investigate whether NFKBIE mutations can affect the proliferation of these cell lines in vitro, we performed competition experiments with mixed cultures of cells with wild type and mutated NFKBIE. Analysis of the clonal composition after 2 weeks showed no change in the mutant allele frequency (MAF), suggesting that NFKBIE mutations do not affect the spontaneous in vitro growth of the immortalized leukemia cells. However, repeated TLR or BCR stimulation of these cells with CpG-DNA, LPS, anti-IgM or autoantigen resulted in a 2-3 fold increase in MAF, suggesting that NFKBIE mutations provide a growth advantage when the cells are exposed to certain microenvironmental signals (n=3 experiments/condition, P<0.05 for each condition).To investigate the impact of NFKBIE mutations on leukemia growth in vivo, the same cells were transplanted by intraperitoneal injection in wild type mouse recipients (n=8) and the clonal composition was determined 3 weeks later by MAF analysis of cells isolated from peritoneal cavity (PC), blood and spleen. A significant increase in MAF was observed only in leukemia cells isolated from the spleen (P<0.05), suggesting that microenvironmental signals that positively select NFKBIE-mutated cells are available only in certain tissue compartments.Because mutations in other NF-kB pathway genes have been associated with resistance to IBR in mantle cell lymphoma, we next investigated whether NFKBIE mutations can also affect the response to IBR treatment. In vitro BrdU-incorporation experiments showed that IBR inhibits the proliferation of cells with mutated NFKBIE to a significantly lesser extent compared to cells with wild type NFKBIE (% proliferating cells with wild type and mutated NFKBIE, respectively, cultured without IBR: 90% vs 88%, P=n.s., with 0.2 μM IBR: 57% vs 73%, P<0.001, with 1.0 μM IBR: 28% vs 53%, P<0.001). Consistent with this finding, positive selection of NFKBIE-mutated cells was observed in the presence of IBR after 14 days in mixed culture competition experiments (mean MAF without IBR 47%, with 0.2 μM IBR 61%, p=0.032, with 1.0 μM IBR 64%, p=0.034). The greater resistance of NFKBIE-mutated cells to IBR was further validated by in vivo competition experiments showing a significantly greater increase in MAF in mice treated with IBR compared to controls in all three investigated compartments (n=4 mice/group, PC: P=0.029, blood P=0.029, spleen: P=0.001).To validate these findings in the clinical setting, we investigated the presence of NFKBIE mutations in a cohort of 84 IBR-treated CLL patients. Mutations of NFKBIE were detected at pre-treatment in 10/84 patients, 7/10 with >10% VAF values. Kaplan Meier analysis showed a trend towards reduced progression-free and overall survival from the beginning of IBR treatment for NFKBIE-mutated cases (Figure 1A). Analysis of an extended cohort of over 200 cases is ongoing and will be presented at the meeting.Finally, to investigate whether leukemic cells with mutated NFKBIE remain sensitive to other BCR inhibitors, we tested their growth in the presence of the PI3K inhibitor idelalisib or SYK inhibitor fostamatinib (Figure 1B). In contrast to IBR, both drugs inhibited the proliferation of NFKBIE-mutated cells in vitro, with a greater effect observed with idelalisib. Collectively, these data demonstrate that NFKBIE mutations can reduce the response to IBR treatment and suggest that such cases may benefit more from treatment with a PI3K inhibitor. [Display omitted] DisclosuresMarasca: Janssen: Honoraria, Other: Travel grants; AstraZeneca: Honoraria; AbbVie: Honoraria, Other: Travel grants. Tafuri: Roche: Research Funding; Novartis: Research Funding; Celgene: Research Funding. Laurenti: Janssen: Consultancy, Honoraria; AstraZeneca: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria, Research Funding; Roche: Honoraria, Research Funding; Gilead: Honoraria; BeiGene: Honoraria. Gattei: abbVie: Research Funding; Janssen: Research Funding; Menarini: Research Funding.

AID overexpression leads to aggressive murine CLL and nonimmunoglobulin mutations that mirror human neoplasms

Most cancers become more dangerous by the outgrowth of malignant subclones with additional DNA mutations that favor proliferation or survival. Using chronic lymphocytic leukemia (CLL), a disease that exemplifies this process and is a model for neoplasms in general, we created transgenic mice overexpressing the enzyme activation-induced deaminase (AID), which has a normal function of inducing DNA mutations in B lymphocytes. AID not only allows normal B lymphocytes to develop more effective immunoglobulin-mediated immunity, but is also able to mutate nonimmunoglobulin genes, predisposing to cancer. In CLL, AID expression correlates with poor prognosis, suggesting a role for this enzyme in disease progression. Nevertheless, direct experimental evidence identifying the specific genes that are mutated by AID and indicating that those genes are associated with disease progression is not available. To address this point, we overexpressed Aicda in a murine model of CLL (Eμ-TCL1). Analyses of TCL1/AID mice demonstrate a role for AID in disease kinetics, CLL cell proliferation, and the development of cancer-related target mutations with canonical AID signatures in nonimmunoglobulin genes. Notably, our mouse models can accumulate mutations in the same genes that are mutated in human cancers. Moreover, some of these mutations occur at homologous positions, leading to identical or chemically similar amino acid substitutions as in human CLL and lymphoma. Together, these findings support a direct link between aberrant AID activity and CLL driver mutations that are then selected for their oncogenic effects, whereby AID promotes aggressiveness in CLL and other B-cell neoplasms.

Telomere Shortening By Terc Knockout in the Eµ-TCL1 Transgenic Murine Model of CLL: Characterization of Disease Development and Survival

In chronic lymphocytic leukemia (CLL) short telomeres are associated with other adverse prognostic factors and poor survival. We and others have described association of telomere length with genomic complexity and clonal evolution in CLL. To understand if telomere shortening rather than being only a marker of cell proliferation, could also functionally contribute to disease progression, we generated Terc knock out in the Eµ-TCL1 murine CLL model (Eµ-TCL1 mTerc-/- mice). Comparison of the Eµ-TCL1 mTerc-/- mice from the generations G1, G2 and G3 with that of Eµ-TCL1 (TCL1+) did not show a difference in disease initiation, progression as well as survival even though a significant decrease in telomere length of tumor cells was observed with increasing generations. Of interest, the Eµ-TCL1 mTerc-/- G3 tumors (n=8; G3) more frequently showed defective DNA damage response compared to TCL1+ tumors (n=8), as analysed by changes in phosphorylation of gamma-H2AX, ATM and p53 measured by FACS at 1, 3, 6, 16 and 24 hours after gamma irradiation. Despite this predisposition to undergo genomic complexity, the G3 mice showed no difference in disease development compared to TCL1+ mice. Therefore we investigated if cell extrinsic factors in Terc-/- mice could affect tumor development. The Terc-/- microenvironment is known to be restrictive to B and T-lymphopoiesis and could hence inhibit CLL development. To study the impact of Terc-/- microenvironment, splenic tumors from the Eµ-TCL1 mTerc-/- G3 (n=11; G3) mice were transferred into syngeneic Terc+/+ C57Bl6 mice and compared with that of TCL1+ (n=11). No significant difference in disease burden and survival was observed between these 2 cohorts, indicating that there is no adverse influence of the Terc-/- microenvironment on the tumor growth in the Eµ-TCL1 mTerc-/- mice. Further, we hypothesized that the telomere length in the G3 mice may not be short enough to induce genomic instability and enhance disease aggressiveness. The mice were thus crossed to obtain the Eµ-TCL1 mTerc-/- generation G4 (G4). Though the survival of these mice were similar to that of Eµ-TCL1 controls (median survival :49 vs. 51 weeks; P=0.301), the G4 mice showed significantly decreased disease burden as measured by spleen weight, liver weights and tumor cell fraction. The G4 mice showed decreased fertility and hence further crosses to generate G5 were not performed. However, further shortening of telomeres was achieved by serially transplanting the tumors from G3 into syngeneic recipient mice for 2 generations. Telomere length of the tumor cells analysed by Q-PCR showed a significant decrease with increasing transfer rounds, compared to primary TCL1+ tumors. Interestingly, the 2nd round transfer of the G3 tumors led to a less severe disease and significantly longer survival of the recipient mice compared to 2nd round TCL1+ tumors transplants (median survival from date of transplantation: 15 vs. 7 weeks; P=0.030), indicating that cell intrinsic factors in the G3 tumors hamper proliferation of these cells. We finally analyzed if absence of telomerase could mask disease aggressiveness of the G3 tumors by crossing the G3 mice with wildtype Terc+/+ mice. The resulting TCL1+ mTerc+/- G4 mice showed an increased telomerase activity but had significantly shorter telomere length compared to TCL1+ mice. Strikingly, the TCL1+ mTerc+/- G4 mice showed faster disease development and significantly shorter survival (median survival: 42 vs. 51 weeks; p&lt;0.001) as compared to TCL1+ mice. The aggressiveness of the TCL1+ mTerc+/- G4 tumors was further verified using adoptive transfer into syngeneic mice, and mice transplanted with these tumors showed a significantly shorter survival as compared to TCL1+ or G3 tumors. In summary, the TCL1+ mTerc-/- mice crossed through generations G1 to G4 did not show a difference in disease initiation, progression or survival despite significant shortening of telomeres. However, the tumors from G3 had defective DDR, indicating a potential for accumulating genetic aberrations and clonal evolution. But the absence of functional telomerase decreased the growth potential of these genomic instable tumors. Reconstitution of telomerase in G3 mice resulted in aggressive tumors with short telomere length and could therefore be a valuable murine model for genomic instability and aggressive CLL. Disclosures Tausch: Roche: Consultancy, Honoraria, Speakers Bureau; AbbVie: Consultancy, Honoraria, Other: travel support, Speakers Bureau. Schneider:Celgene: Other: travel grant. Döhner:AbbVie, Agios, Amgen, Astellas, Astex, Celator, Janssen, Jazz, Seattle Genetics: Consultancy, Honoraria; AROG, Bristol Myers Squibb, Pfizer: Research Funding; Celgene, Novartis, Sunesis: Honoraria, Research Funding. Stilgenbauer:AbbVie, AstraZeneca, Celgene, Gilead Sciences, Inc., GSK, Hoffmann La-Roche, Janssen, Novartis: Consultancy, Honoraria, Research Funding, Speakers Bureau.

Oxidative stress as candidate therapeutic target to overcome microenvironmental protection of CLL.

Chronic lymphocytic leukemia (CLL) cells depend on microenvironmental non-malignant cells for survival. We compared the transcriptomes of primary CLL cells cocultured or not with protective bone marrow stromal cells (BMSCs) and found that oxidative phosphorylation, mitochondrial function, and hypoxic signaling undergo most significant dysregulation in non-protected CLL cells, with the changes peaking at 6-8 h, directly before induction of apoptosis. A subset of CLL patients displayed a gene expression signature resembling that of cocultured CLL cells and had significantly worse progression-free and overall survival. To identify drugs blocking BMSC-mediated support, we compared the relevant transcriptomic changes to the Connectivity Map database. Correlation was found with the transcriptomic signatures of the cardiac glycoside ouabain and of the ipecac alkaloids emetine and cephaeline. These compounds were highly active against protected primary CLL cells (relative IC50's 287, 190, and 35 nM, respectively) and acted by repressing HIF-1α and disturbing intracellular redox homeostasis. We tested emetine in a murine model of CLL and observed decreased CLL cells in peripheral blood, spleen, and bone marrow, recovery of hematological parameters and doubling of median survival (31.5 vs. 15 days, P = 0.0001). Pathways regulating redox homeostasis are thus therapeutically targetable mediators of microenvironmental support in CLL cells.

PF356 CHRONIC LYMPHOCYTIC LEUKEMIA INDUCES SKEWING OF (ANTIGEN‐SPECIFIC) T CELL RESPONSES TOWARDS A DYSFUNCTIONAL SHORT‐LIVED EFFECTOR PHENOTYPE

Background:In recent years, CAR T cells showed to be highly effective in B cell malignancies such as acute lymphocytic leukemia. However, for chronic lymphocytic leukemia (CLL) efficacy of autologous T cell therapies is rather limited. T cells of CLL patients have a dysfunctional, terminally differentiated phenotype, which might be linked with the lack of success of T cell therapies. Recently it was shown that presence of memory T cell subsets in the CAR T cell infusion product associated with complete responses in CLL (Fraietta et al., 2018).Aims:It remains to be elucidated how CLL cells influence T cell skewing and whether this affects T cell responses in vivo. In this context, we functionally investigated acute T cell responses in the TCL1 mouse model, which features similar T cell dysfunction as observed in CLL patients (Gorgun et al., 2009). We applied a set‐up that allows to study the response against acute infection of (1) endogenous CD8+ T cells, which have been exposed to leukemic cells during CLL development, and (2) the antigen‐specific response of naïve, transferred OT‐I T cells.Methods:C57BL/6J (CD45.2+) mice were injected i.p. with 20x106 TCL1 splenocytes or PBS. When CD5+ leukemia reached 70% cells in blood, 50.000 naïve ovalbumin‐specific CD8+ T cells (OT‐I, CD45.1+) were injected i.v. Next, all mice were infected with mCMV‐OVA and sacrificed 7 days later. Splenocytes were analyzed directly or after in vitro ovalbumin stimulation by FACS.Results:(1) Already prior to infection, we found a skewing towards antigen‐experienced cells in the endogenous CD8+ compartment of leukemic mice. After mCMV‐OVA infection, a population of naïve CD8+ T cells persisted in control mice, but in leukemic mice the complete CD8+ compartment differentiated into effector cells. Within the effector compartment, we found a skewing to the short‐lived effector cell (SLEC) phenotype in the leukemic mice, while memory precursor effector cells (MPEC) were reduced. This skewing towards SLEC associated with increased effector‐associated T‐bet expression.(2) Analysis of the OT‐I cells allowed to perform detailed study of the impact of leukemia on an antigen‐specific response. Seven days after infection, spleens of leukemic and control mice contained similar frequencies of differentiated OT‐I cells, that had an effector phenotype. Also here, TCL1‐derived OT‐I cells showed a decreased percentage of MPEC, while SLECs were increased (Figure 1), indicating impaired memory formation. In correlation with skewing towards SLECs in leukemic mice, these OT‐I cells showed reduced expression of memory‐associated transcription factor Bcl6, while T‐bet was also increased. Earlier it was shown that Bcl6 is able to repress glycolysis, essential for T cell effector function (Oestreich et al., 2014). In line with reduced Bcl6 expression, we found increased glucose uptake in TCL1‐derived OT‐I cells. Despite the skewing towards SLEC phenotype, the TCL1‐derived OT‐I cells showed decreased production of IFNγ and TNFα and decreased degranulation (CD107a expression) upon in vitro restimulation.Summary/Conclusion:In this murine model of CLL, both bystander T cells as well as acute antigen‐specific T cell responses are driven towards an SLEC phenotype with signs of functional impairment, and metabolic alterations. This approach may provide crucial insight into the interplay between CLL and T cells, and the underlying failure of cancer immune surveillance.image

A Murine Model of Chronic Lymphocytic Leukemia Based on B Cell-Restricted Expression of Sf3b1 Mutation and Atm Deletion

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

STAT3 mediates C6-ceramide-induced cell death in chronic lymphocytic leukemia

The pathogenesis of chronic lymphocytic leukemia (CLL) is poorly understood and it remains incurable with current therapies. We have previously shown that nanoliposomal C6-ceramide (CNL) is an effective therapy in an in vivo murine model of CLL. However, the key signaling pathways mediating CNL-induced cell death in CLL remains unknown. We hypothesized that CNL targets STAT3, a critical regulator of hematopoietic biology. We observed that CNL treatment reduced phosphorylated STAT3 at both Y705 and S727 residues in CLL cell lines and patient cells. This, in turn, reduced STAT3 transcriptional activity and expression of critical STAT3-dependent survival factors like Mcl-1 and survivin. The effect of CNL on STAT3 was further confirmed ex vivo as shown by reduced STAT3 phosphorylation in xenograft tumors obtained from mice treated with CNL. CNL suppressed STAT3 phosphorylation at Y705 and S727 through reduction in BTK activity and MEK1/2 kinase/PKC activities, respectively. Moreover, a synergistic reduction in CLL cell viability was observed on co-treatment with CNL and the BTK inhibitor, ibrutinib. Expression of an oncogenic form of STAT3 conferred partial resistance to CNL, providing confirmation that STAT3 mediates CNL-induced cell death. Taken together, these findings provide the first body of evidence demonstrating ceramide regulation of STAT3 phosphorylation. These results are also the first to demonstrate an effect of ceramide on BTK, a critical kinase mediating the B-cell receptor signaling in CLL cells and suggest a novel and synergistic combination of CNL and BTK inhibitors for CLL treatment.

Combined BTK and PI3Kδ Inhibition with Acalabrutinib and ACP-319 Improves Survival and Tumor Control in CLL Mouse Model.

Purpose: Targeting the B-cell receptor (BCR) pathway with inhibitors of Bruton tyrosine kinase (BTK) and PI3Kδ is highly effective for the treatment of chronic lymphocytic leukemia (CLL). However, deep remissions are uncommon, and drug resistance with single-agent therapy can occur. In vitro studies support the effectiveness of combing PI3Kδ and BTK inhibitors.Experimental Design: As CLL proliferation and survival depends on the microenvironment, we used murine models to assess the efficacy of the BTK inhibitor acalabrutinib combined with the PI3Kδ inhibitor ACP-319 in vivo We compared single-agent with combination therapy in TCL1-192 cell-injected mice, a model of aggressive CLL.Results: We found significantly larger reductions in tumor burden in the peripheral blood and spleen of combination-treated mice. Although single-agent therapy improved survival compared with control mice by a few days, combination therapy extended survival by over 2 weeks compared with either single agent. The combination reduced tumor proliferation, NF-κB signaling, and expression of BCL-xL and MCL-1 more potently than single-agent therapy.Conclusions: The combination of acalabrutinib and ACP-319 was superior to single-agent treatment in a murine CLL model, warranting further investigation of this combination in clinical studies. Clin Cancer Res; 23(19); 5814-23. ©2017 AACR.

Leukemic cell expressed CTLA-4 suppresses T cells via down-modulation of CD80 by trans-endocytosis

Abstract Despite clinical utility in targeting the immune checkpoint, Cytotoxic T Lymphocyte Antigen 4 (CTLA-4), on T cells, its function on non-T cells remains unaddressed, especially in the context of therapy. We define an immunosuppressive role for tumor expressed CTLA-4 in Chronic Lymphocytic Leukemia (CLL). Most primary CLL samples were intracellularly CTLA-4+ (12/14) but surface CTLA-4− (14/14). Co-culture with activated T cells induced surface expression of CTLA-4 on CLL B cells. To mechanistically study CLL B cell expressed CTLA-4, we generated CLL-derived Mec1 and OSU-CLL cell lines to inducibly express CTLA-4. These cell lines express the ligands for CTLA-4, CD80 and CD86. Expression of CTLA-4 on Mec1 and OSU-CLL resulted in decreased CD80 expression on CD80+ cells with rescue upon CTLA-4 blockade (N=3). Co-culture of CTLA-4+ Mec1 and CTLA-4+ primary CLL cells with CD80-GFP+ Hek293 cells revealed transfer of CD80-GFP into primary CLL cells (N=2) and the Mec1 cell line. This finding was consistent with the ability of CTLA-4+ T cells to trans-endocytose CD80. Co-culture of T cells with Mec1 CTLA-4+ cells resulted in decreased IL2 production (N=3, p=0.0172). The loss of IL2 signified decreased co-stimulation as a result of tumor expressed CTLA-4. We generated an in vivo model to study tumor CTLA-4 using the TCL1 murine model of CLL. Murine CTLA-4+ (mCTLA-4) TCL1 tumor cells were engrafted into human CTLA-4+/+mFcγR−/− immune competent mice. MCTLA-4 on the tumor cells but not human CTLA-4 on the host cells was blocked with an anti-mCTLA-4 antibody. Effect on disease progression and overall survival are currently being assessed. We present a paradigm shift whereby CTLA-4 is an immunosuppressive protein irrespective of expression on T cells.

Splenic Marginal Zone Granulocytes Acquire an Accentuated Neutrophil B-Cell Helper Phenotype in Chronic Lymphocytic Leukemia.

Recruitment of tumor-associated macrophages and neutrophils (TAM and TAN) to solid tumors contributes to immunosuppression in the tumor microenvironment; however, their contributions to lymphoid neoplasms are less clear. In human chronic lymphocytic leukemia (CLL), tumor B cells lodge in lymph nodes where interactions with the microenvironment occur. Tumor cell homing stimulates proliferation, such that engagement of the B-cell receptor is important for malignant progression. In the Eμ-Tcl1 murine model of CLL, we identified gene expression signatures indicative of a skewed polarization in the phenotype of monocytes and neutrophils. Selective ablation of either of these cell populations in mice delayed leukemia growth. Despite tumor infiltration of these immune cells, a systemic inflammation was not detected. Notably, in progressive CLL, splenic neutrophils were observed to differentiate toward a B-cell helper phenotype, a process promoted by the induction of leukemia-associated IL10 and TGFβ. Our results suggest that targeting aberrant neutrophil differentiation and restoring myeloid cell homeostasis could limit the formation of survival niches for CLL cells. Cancer Res; 76(18); 5253-65. ©2016 AACR.

The PI3K/mTOR inhibitor PF-04691502 induces apoptosis and inhibits microenvironmental signaling in CLL and the Eµ-TCL1 mouse model

AbstractCurrent treatment strategies for chronic lymphocytic leukemia (CLL) involve a combination of conventional chemotherapeutics, monoclonal antibodies, and targeted signaling inhibitors. However, CLL remains largely incurable, with drug resistance and treatment relapse a common occurrence, leading to the search for novel treatments. Mechanistic target of rapamycin (mTOR)-specific inhibitors have been previously assessed but their efficacy is limited due to a positive feedback loop via mTOR complex 2 (mTORC2), resulting in activation of prosurvival signaling. In this study, we show that the dual phosphatidylinositol 3-kinase (PI3K)/mTOR inhibitor PF-04691502 does not induce an mTORC2 positive feedback loop similar to other PI3K inhibitors but does induce substantial antitumor effects. PF-04691502 significantly reduced survival coincident with the induction of Noxa and Puma, independently of immunoglobulin heavy chain variable region mutational status, CD38, and ZAP-70 expression. PF-04691502 inhibited both anti–immunoglobulin M–induced signaling and overcame stroma-induced survival signals and migratory stimuli from CXCL12. Equivalent in vitro activity was seen in the Eμ-TCL1 murine model of CLL. In vivo, PF-04691502 treatment of tumor-bearing animals resulted in a transient lymphocytosis, followed by a clear reduction in tumor in the blood, bone marrow, spleen, and lymph nodes. These data indicate that PF-04691502 or other dual PI3K/mTOR inhibitors in development may prove efficacious for the treatment of CLL, increasing our armamentarium to successfully manage this disease.

Immune Checkpoint Blockade with Anti-PD-L1 Prevents Immune Dysfuntion and CLL Development in the TCL1 Adoptive Transfer Mouse Model

Background: Clinical studies have demonstrated that targeted immunotherapy using PD-1/PD-L1 antibodies induces tumor regression and prolongs disease stabilization in advanced solid cancers. Data on the clinical efficacy in hematological malignancies is largely missing, even though PD-L1/PD-1 interactions have been described as major mediators of immune dysfunction in several leukemias and lymphomas. They are therefore ideal to study if PD-L1/PD-1 blockade has the potential to control disease by restoring anti-tumor immune responses. Several groups showed that chronic lymphocytic leukemia (CLL) provokes immune evasion via PD-L1/PD-1 inhibitory signaling, and that this is very closely mirrored in the Eµ-TCL1 (TCL1) murine model for CLL. Our recent data suggest that in this model, aberrant PD-L1 expression in myeloid cells contributes to the immune defect in CLL. We further demonstrated that the T cell and myeloid cell immune defects in ageing leukemic mice can be induced in young wild-type (WT) mice by adoptive transfer (AT) of murine CLL. In the current study, we used the AT model to test if in vivoPD-L1 blockade corrects leukemia-induced cellular immune dysfunction in myeloid and T cells and enhances anti-tumor immunity.Methods: WT mice transplanted with 4x107 TCL1 splenocytes were randomized to treatment with 10 mg/kg α-murine-PD-L1 (n=15) or isotype antibody (n=10), which was administered i.p. every 3 days starting 1 day after AT, and sacrificed 31 days later. Matched non-transplanted WT mice (n=6) served as additional controls. Immune cell subsets, expression of immune checkpoint markers and T cell effector functions were analyzed by multicolor flow cytometry using cells isolated form spleen, peripheral blood (PB), bone marrow (BM) and peritoneal cavity (PC). Cell proliferation was measured by EdU incorporation in vivo. Immune synapse (IS) formation was assessed by confocal microscopy. Serum cytokines were quantified by multiplex bead arrays.Results: We first confirmed successful engraftment and presence of disease by immunohistochemistry. Compared to isotype controls, α-PD-L1 treated mice had significantly lower spleen weights (median 0.2 g vs 0.9 g, p<0.0001) and a highly significant lower relative frequency of CD19+CD5+ CLL lymphocytes in spleen (1.55% vs 71.69%), PB (10.5% vs 63.53%) and BM (0.26% vs 2.74%) demonstrating very effective tumor control. Compared to non-transplanted animals, α-PD-L1 treated mice showed alterations in almost all phenotypical and functional immune cell parameters, especially in regards to immune cell activation, indicating encounter with and immunological challenge by CLL cells. Along with disease control, α-PD-L1 treated mice had improved immune status as multiple inflammatory cytokines in the serum, including IL-10, TNF-α, CCL2 and GM-CSF were decreased and splenic infiltration of monocytes was reduced. While CLL development skewed monocytes towards Ly6Clow patrolling monocytes, α-PD-L1 treatment restored the presence of Ly6Chi inflammatory monocytes and decreased the expression of adhesion molecules ICAM-1 and PECAM-1. These monocytes regained their differentiation capacity as shown by increased numbers of macrophages and mature MHC-IIhi dendritic cells in the spleens of treated mice. In the T cell compartment, in vivo PD-L1 blockade prevented the CLL-induced CD4/CD8 ratio inversion, the loss of naïve CD8 T cells and the shift towards antigen-experienced and terminally differentiated T cells in spleen, BM and PB. Aberrant expression of immune checkpoint markers PD-1, KLRG-1, LAG-3, and 2B4 was also significantly reduced. The CLL-associated loss of intracellular IL-2 and the increased secretion of IL-4 and IFN-γ in CD4 T cells were prevented in α-PD-L1 treated mice. Respective cytokine patterns were observed in the serum. Functionally, PD-L1 blockade restored CD8 degranulation and IS formation to the level of healthy T cells, and significantly improved both ex vivo and in vivoT cell proliferation.Conclusion: Our in vivodata demonstrate that early PD-L1 blockade very effectively controls CLL development and enables complex effector function of myeloid and T cells, thus restoring anti-tumor immune responses. Targeting PD-L1/PD-1 interactions should therefore be further explored in clinical studies, potentially in combination with novel substances.BH/FM and MS/JGG contributed equally to first and last authorship. DisclosuresNo relevant conflicts of interest to declare.

Absence of Caveloin-1 Leads to Delayed Development of Chronic Lymphocytic Leukemia in Eµ-TCL1 Mouse Model

Background: Chronic Lymphocytic Leukemia (CLL) is the most common adult leukemia in the United States. Clinical heterogeneity, a characteristic feature of CLL is a major problem in the clinical management of this currently incurable leukemia. We and others have demonstrated that the tissue microenvironment, specifically the lymph node (LN), influence the biological and clinical behavior including the clinical heterogeneity of CLL. Using gene expression profiling of CLL cells from peripheral blood (PB), bone marrow (BM) and LNs, we identified Cav-1 a member of the Tolerogenic Signature (genes associated with host immune tolerance) as one of the candidate genes which might be involved in the pathogenesis of CLL. We found that Cav-1 levels were significantly elevated (11 fold) in CLL cells from LNs compared to BM and PB. Cav-1 is the major element of caveolae, which are flask-shaped membrane invaginations. Cav-1 is involved in multiple cellular processes like the regulation and transportation of cellular cholesterol and lipids, clathrin independent endocytosis and signal transduction leading to oncogenesis or tumor suppression. We have previously shown that knock down of Cav-1 results in a significant decrease in cell migration and proliferation of primary human CLL cells in vitro. We have also demonstrated that knock down of Cav-1 prevents CLL cells from forming immune synapses. These immune synapses are important for the interaction between the CLL cells and their tumor microenvironment. These results suggest that Cav-1 protect CLL cells from undergoing apoptosis and enhances their migration in vitro.Objectives and Methodology: To understand the precise role of Cav-1 in leukemic progression in vivo, we crossed Cav-1-/- mice to Eµ-TCL1 mice, which is a well-established transgenic murine model for CLL. The offspring were observed and evaluated for the development of CLL. These mice were sacrificed at the age of 12, 24, 36 and 40+ weeks and peripheral blood, bone marrow and spleen and were examined for the presence of CD5+B220+CD19+ CLL cells using flow cytometry. Spleen, lymph nodes, liver, lungs and kidney were evaluated for the presence of CLL cells using H&E staining of histologic slides.Results: To study the role of Cav-1 in Eµ-TCL1, we isolated splenic B cells and measured the expression of Cav-1. We observed a gradual increase in the expression of Cav-1 in splenic B cells from Eµ-TCL1 mice at age of 12, 24 and 36 weeks when compared with wild type mice. This suggested that Cav-1 might be playing a role in CLL progression in Eµ-TCL1 mice. Therefore, to study the role of Cav-1 in CLL disease progression we decreased the expression of Cav-1 in vivo by breeding Eµ-TCL1 with Cav1 knockout mice. We generated Eµ-TCL1-Cav1-/+ and Eµ-TCL1-Cav1-/- mice to study the effect of Cav-1 knock down in aggressiveness of CLL in vivo. We have shown that Cav-1 is overexpressed in CLL cells from patients with poorer clinical outcome and protects CLL cells from undergoing apoptosis. Therefore, we analyze the number of CLL cells in Eµ-TCL1-Cav1-/+ and Eµ-TCL1-Cav1-/- mice. We observed a significant reduction in the number of B220+CD5+ CLL cells population in bone marrow and spleen of Eµ-TCL1-Cav1-/+ and Eµ-TCL1-Cav1-/- mice when compared with Eµ-TCL1-Cav1wt/wt mice. We have previously shown that Cav-1 is important for CLL cells migration in vitro. Therefore, to study its effect in vivo we analyzed infiltration of CLL cells in spleen, lymph nodes, liver, kidney and lungs in these mice. There was no or significant decrease in tumor infiltration of CLL cells in spleen, lymph nodes, liver, lungs and kidney in Eµ-TCL1-Cav1-/+ and Eµ-TCL1-Cav1-/- mice when compared with Eµ-TCL1-Cav1wt/wt alone. Next, we wanted to examine the effect of Cav-1 knock down on splenomegaly and hepatomegaly. We found that there was a significant decrease in splenomegaly and hepatomegaly in Eµ-TCL1-Cav1-/+ and Eµ-TCL1-Cav1-/- mice. The spleen and liver size of Eµ-TCL1-Cav1-/+ and Eµ-TCL1-Cav1-/- mice was significantly reduced when compared with Eµ-TCL1 mice. Together these results suggest that high expression of Cav-1 in CLL cells leads to enhance proliferation and promotes disease progression in Eµ-TCL1 mice. DisclosuresNo relevant conflicts of interest to declare.

STAT3 Mediates C6-Ceramide-Induced Cell Death in Chronic Lymphocytic Leukemia

Chronic lymphocytic leukemia (CLL) is the most prevalent form of leukemia in adults in Western countries. Despite high incidence, its pathogenesis is still poorly understood and it remains incurable with current therapies. Ceramide is a ‘tumor suppressor’ lipid known to regulate differentiation, senescence and cell cycle arrest. Previous work from our laboratory has demonstrated the therapeutic efficacy of nanoliposomal C6-ceramide in several solid and non-solid tumor models. We have shown that nanoliposomal C6-ceramide selectively induces cell death in CLL cells by targeting the Warburg effect with no detrimental effects on normal peripheral blood mononuclear cells. Additionally, nanoliposomal C6-ceramide treatment results in tumor regression in an in vivo murine model of CLL. However, the mechanism and key molecular players mediating C6-ceramide-induced cell death in CLL remains unknown. Constitutive activation of signal transducer and activator of transcription 3 (STAT3) has been implicated in many cancers. Several reports in the literature have shown that STAT3 is constitutively phosphorylated on serine 727 (S727 p-STAT3) in CLL and STAT3 might be a therapeutic target in this disease. We hypothesize that STAT3 is a target of C6-ceramide in CLL cells.We first validated STAT3 as a therapeutic target in CLL. STAT3 knockdown in CLL cells induced cell death. C6-ceramide treatment also induces necrotic cell death in these cells. Examination of STAT3 phosphorylation state demonstrates constitutive phosphorylation at S727 and Y705 residue in CLL cell lines and patient cells. C6-ceramide treatment suppressed the levels of Y705 p-STAT3 and S727 p-STAT3, while having no effect on total STAT3 levels. The downregulation of phosphorylation was specific to STAT3 and did not affect the other STATs. C6-ceramide treatment also led to the downregulation of several downstream targets of STAT3 like p21, cyclin D1, Mcl-1 and survivin. Furthermore, the suppression in STAT3 phosphorylation preceded induction of C6-ceramide-mediated cell death in CLL cells, suggesting that this event might be critical for cell death induction. Analysis of the upstream kinases revealed that C6-ceramide-induced suppression of S727 p-STAT3 might be, in part, a consequence of suppression of MEK1/2 kinase and protein kinase C (PKC). Ongoing studies will focus on identifying the specific phosphorylation site(s) that mediates cell death and examine the effect of C6-ceramide on upstream regulators of p-STAT3 in an effort to delineate the molecular mechanism of C6-ceramide-induced cell death in CLL. In conclusion, our data suggests that targeting STAT3 with nanoliposomal C6-ceramide is a potential therapy for CLL. This work is the first body of evidence demonstrating an effect of C6-ceramide on STAT3 phosphorylation. [Display omitted] DisclosuresKester:Keystone Nano, Inc: Employment, Equity Ownership.

The Dual PI3K/mTOR Inhibitor PF-04691502 Induces Substantial Apoptosis in Chronic Lymphocytic Leukemia Cells in Vitro and Prolongs Survival in the Eµ-TCL1 Mouse Model

Pharmacological inhibition of specific B cell receptor signalling pathways within chronic lymphocytic leukemia (CLL) cells offers the potential for improved therapeutic options with reduced off target toxicity. Idelalisib, the PI3Kδ selective inhibitor, has been approved for CLL and significantly improved overall survival among patients with relapsed CLL in combination with rituximab compared to rituximab alone. In addition to PI3Kδ however, there are three other Class I PI3K isoforms, PI3Kα, PI3Kβ and PI3Kγ, with PI3Kα known to have a role in CLL survival and chemotaxis. In neutrophils, functional redundancy between PI3K isoforms is evident, with inhibition of at least three PI3K isoforms required for maximal apoptosis. Inhibition of mTOR is known to induce cell cycle arrest and apoptosis in CLL cells, however prolonged inhibition of mTOR results in activation of a positive feedback loop resulting in PI3K\\Akt reactivation. To overcome these caveats, pan PI3K inhibition alongside mTOR inhibition may achieve superior cytotoxicity against CLL cells compared to PI3Kδ or mTOR inhibition alone. We therefore sought to investigate the effect of a dual pan PI3K and mTOR inhibitor, PF-04691502, on primary CLL cells and in the Eµ-TCL1 mouse model of CLL.Twenty five primary CLL samples were treated with PF-04691502 and downstream signalling and subsequent apoptosis assessed by immunoblotting and Annexin V/PI staining respectively. In primary CLL cells, PF-04691502 induced 80% apoptosis after 24 hours at 10µM concentration. PF-04691502 had an IC50 value of 1µM as assessed by Annexin V/PI staining, with minimal toxicity to normal human B or T cells and a trend towards more specific killing in unmutated CLL (p=0.09) compared to mutated CLL. PF-04691502 inhibited both soluble and immobilised anti-IgM induced signalling and overcame anti-IgM induced survival signals. PF-04691502 abrogated CXCL12 induced signalling and impaired subsequent CLL migration towards the chemokine in a transwell chemotaxis assay. Finally, PF-04691502 was able to overcome protection from co-culture with stroma inducing significant apoptosis of CLL cells when added continuously and in wash out experiments.To assess the effect of PF-04691502 in vivo, twenty Eµ-TCL1 mice were inoculated with tumour cells and after day 21 dosed once daily with vehicle, 5mg/kg or 10mg/kg PF-04691502 for a further 14 days. Mice were assessed for leukemic cell counts, spleen size and white blood cell count throughout the duration of the experiment. The effect of PF-04691502 on B cell receptor and chemokine receptor induced signalling in ex vivo Eµ-TCL1 cells was assessed by immunoblotting.Near identical results to the CLL data above were observed. Using Eµ-TCL1 cells isolated from the spleen, PF-04691502 was shown to profoundly inhibit anti-IgM and CXCL12 induced signalling and chemotaxis as well as induce substantial apoptosis as measured by Annexin V/PI staining. Therefore, we subsequently assessed the effect of PF-04691502 in vivo using this model. Mice treated with PF-04691502 displayed a transient increase in leukemic cell numbers in the blood persisting for 1-4 days, followed by a reduction to levels significantly below that of the vehicle control. The whole white blood cell count remained stable in the PF-04691502 treated mice during the course of treatment, whilst the vehicle treated mice showed significant increases in tumour load up to 40×106 cells/ml 33 days post inoculation. Spleen sizes increased steadily over time in the control mice, whilst PF-04691502 treatment prevented this. After 33 days, PF-04691502 treated mice had spleen sizes comparable to non tumor-recipient control animals. In addition, CD5+ B220+ Eµ-TCL1 cells were significantly reduced in the bone marrow, spleen and lymph nodes following PF-04691502 treatment compared to control mice (p=0.0198; p< 0.0001; p=0.0151 respectively). These results demonstrate that PF-04691502 induces substantial apoptosis of primary CLL cells in vitro and significantly prolongs survival in an in vivo murine model of CLL. Such data indicate that dual pan PI3K/mTOR inhibitors may prove efficacious in the treatment of CLL patients. DisclosuresNo relevant conflicts of interest to declare.

The CD37-targeted antibody–drug conjugate IMGN529 is highly active against human CLL and in a novel CD37 transgenic murine leukemia model

Therapeutic regimens for chronic lymphocytic leukemia (CLL) have increasingly utilized monoclonal antibodies since the chimeric anti-CD20 antibody rituximab was introduced. Despite improved clinical outcomes, current CLL therapies are not curative. Therefore, antibodies with greater efficacy and novel targets are desirable. One promising target is CD37, a tetraspanin protein highly expressed on malignant B-cells in CLL and non-Hodgkin lymphoma. While several novel CD37-directed therapeutics are emerging, detailed preclinical evaluation of these agents is limited by lack of appropriate animal models with spontaneous leukemia expressing the human CD37 (hCD37) target. To address this, we generated a murine CLL model that develops transplantable hCD37+ leukemia. Subsequently, we engrafted healthy mice with this leukemia to evaluate IMGN529, a novel hCD37-targeting antibody-drug conjugate. IMGN529 rapidly eliminated peripheral blood leukemia and improved overall survival. In contrast, the antibody component of IMGN529 could not alter disease course despite exhibiting substantial in vitro cytotoxicity. Furthermore, IMGN529 is directly cytotoxic to human CLL in vitro, depletes B-cells in patient whole blood, and promotes killing by macrophages and NK cells. Our results demonstrate the utility of a novel mouse model for evaluating anti-human CD37 therapeutics and highlight the potential of IMGN529 for treatment of CLL and other CD37-positive B-cell malignancies.

C6-Ceramide Nanoliposomes Target the Warburg Effect in Chronic Lymphocytic Leukemia

Ceramide is a sphingolipid metabolite that induces cancer cell death. When C6-ceramide is encapsulated in a nanoliposome bilayer formulation, cell death is selectively induced in tumor models. However, the mechanism underlying this selectivity is unknown. As most tumors exhibit a preferential switch to glycolysis, as described in the “Warburg effect”, we hypothesize that ceramide nanoliposomes selectively target this glycolytic pathway in cancer. We utilize chronic lymphocytic leukemia (CLL) as a cancer model, which has an increased dependency on glycolysis. In CLL cells, we demonstrate that C6-ceramide nanoliposomes, but not control nanoliposomes, induce caspase 3/7-independent necrotic cell death. Nanoliposomal ceramide inhibits both the RNA and protein expression of GAPDH, an enzyme in the glycolytic pathway, which is overexpressed in CLL. To confirm that ceramide targets GAPDH, we demonstrate that downregulation of GAPDH potentiates the decrease in ATP after ceramide treatment and exogenous pyruvate treatment as well as GAPDH overexpression partially rescues ceramide-induced necrosis. Finally, an in vivo murine model of CLL shows that nanoliposomal C6-ceramide treatment elicits tumor regression, concomitant with GAPDH downregulation. We conclude that selective inhibition of the glycolytic pathway in CLL cells with nanoliposomal C6-ceramide could potentially be an effective therapy for leukemia by targeting the Warburg effect.

Therapeutic implications of activation of the host gene (Dleu2) promoter for miR-15a/16-1 in chronic lymphocytic leukemia

Genetic lesions and other regulatory events lead to silencing of the 13q14 locus in a majority of chronic lymphocytic leukemia (CLL) patients. This locus encodes a pair of critical pro-apoptotic microRNAs, miR-15a/16-1. Decreased levels of miR-15a/16-1 are critical for the increased survival exhibited by CLL cells. Similarly, in a de novo murine model of CLL, the NZB strain, germline-encoded regulation of the syntenic region resulted in decreased miR-15a/16-1. In this paper we have identified additional molecular mechanisms regulating miR-15a/16-1 levels and shown that the transcription factor BSAP (B cell Specific Activator Protein) directly interacts with Dleu2, the host gene containing the mir-15a/16-1 loci and via negative regulation of the Dleu2 promoter results in repression of mir-15a/16 expression. CLL patient B cell expression levels of BSAP were increased compared to control sources of B cells. With the use of siRNA mediated repression, the levels of BSAP were decreased in vitro in the NZB derived malignant B1 cell line, LNC, and in ex vivo CLL patient PBMC. BSAP knockdown led to an increase in the expression of miR-15a/16-1 and an increase in apoptosis and a cell cycle arrest in both the cell line and patient PBMC. Moreover, using Dleu2 promoter analysis by chromatin immunoprecipitation (ChIP) assay we have shown that BSAP directly interacts with the Dleu2 promoter. Derepression of the Dleu2 promoter via inhibition of histone deacetylation combined with BSAP knockdown increased miR-15a/16 expression and increased malignant B cell death. In summary, therapy targeting enhanced host gene Dleu2 transcription may augment CLL therapy.

RhoH is critical for cell-microenvironment interactions in chronic lymphocytic leukemia in mice and humans

AbstractTrafficking of B-cell chronic lymphocytic leukemia (CLL) cells to the bone marrow and interaction with supporting stromal cells mediates important survival and proliferation signals. Previous studies have demonstrated that deletion of Rhoh led to a delayed disease onset in a murine model of CLL. Here we assessed the impact of RhoH on homing, migration, and cell-contact dependent interactions of CLL cells. Rhoh−/− CLL cells exhibited reduced marrow homing and subsequent engraftment. In vitro migration toward the chemokines CXCL12 and CXCL13 and cell-cell interactions between Rhoh−/− CLL cells and the supporting microenvironment was reduced. In the absence of RhoH the distribution of phosphorylated focal adhesion kinase, a protein known to coordinate activation of the Rho GTPases RhoA and Rac, appeared less polarized in chemokine-stimulated Rhoh−/− CLL cells, and activation and localization of RhoA and Rac was dysregulated leading to defective integrin function. These findings in the Rhoh−/− CLL cells were subsequently demonstrated to closely resemble changes in GTPase activation observed in human CLL samples after in vitro and in vivo treatment with lenalidomide, an agent with known influence on microenvironment protection, and suggest that RhoH plays a critical role in prosurvival CLL cell-cell and cell-microenvironment interactions with this agent.

Development of CLL in the TCL1 transgenic mouse model is associated with severe skewing of the T-cell compartment homologous to human CLL

Chronic lymphocytic leukemia (CLL) cells require complex microenvironmental and immunologic interactions to survive and proliferate. Such interactions might be best recreated in animal models; however, this needs extensive verification. We therefore investigated the composition of the T-cell compartment in the Eμ-TCL1 transgenic mouse, currently the most widely used murine model for CLL. Immunophenotyping and transplant approaches were used to define T-cell subsets at various stages of CLL. Analogous to human CLL, we observed a skewing of T-cell subsets from naive to antigen-experienced memory T cells that was more pronounced in lymph nodes than in blood. Transplantation of CLL into non-transgenic recipients was feasible without immunosuppression in a pure C57BL/6 background and resulted in the prominent skewing of the T cells of the recipient mice. Both in spontaneously developed CLL and in the transplantation setting, a loss in T-cell receptor diversity was observed, with a relevant number of clonal T-cell populations arising. This suggests that antigen-dependent differentiation toward the T memory pool is initiated by murine CLL cells. In summary, we validate the TCL1 transgenic mouse model for analysis of T-cell phenotypes and suggest a CLL-dependent antigen-driven skewing of T cells in these mice.