Human Chronic Lymphocytic Leukemia Research Articles

BCL3 Expression Is a Potential Prognostic and Predictive Biomarker in Acute Myeloid Leukemia of FAB Subtype M2

Although the implication of BCL3 has been disclosed in human chronic lymphocytic leukemia as well as other solid tumors, the diagnostic and prognostic of BCL3 expression in acute myeloid leukemia (AML) remains largely unclear. In this study, we isolated total RNA from bone marrow mononuclear cells collected from 101 de novo AML patients and 27 healthy donors. After reverse transcription, quantitative real-time PCR was performed to detect BCL3 expression level. BCL3 mRNA level was significantly down-regulated in BMMCs of AML patients compared with healthy controls (P = 0.0015). BCL3 was showed a higher level in AML patients with poor-risk karyotypes than that of in patients with favorable/intermediate-risk karyotypes (P = 0.014). ROC analysis demonstrated that BCL3 could effectively differentiate AML patients from normal controls. Among the French-American-British (FAB) subtypes, the frequency of low BCL3 expression in M2 subtypes is significantly higher than that of in the other subtypes M1/M4/M5/M6/M7 (P = 0.006), and mildly lower in myelomonocytic/monocytic subtypes M4/M5 (P = 0.064) than those in M1/M2/M6/M7 subtypes. Chromosome analysis revealed that BCL3low patients had a remarkably higher frequency of t (8;21) abnormality (P = 0.0047) and lower frequency of normal karyotype (P = 0.0059) than BCL3high patients. BCL3high patients showed a significantly higher frequency of FLT3-ITD mutation (P = 0.028) and lower frequency of C-Kit mutation (P = 0.0232) than BCL3low patients. Although there were no significant differences in complete remission and overall survival between BCL3low and BCL3high groups, patients with high BCL3 expression markedly shorter overall survival (OS, P = 0.049), relapse-free survival (RFS, P = 0.027) and disease-free survival (DFS, P = 0.042) in M2 AML than low BCL3 expression patients. Additionally, in AMLs of M2 subtype, high BCL3 expression patients had markedly lower complete remission (CR) rate (P = 0.0317) after the second induction treatment than patients with BCL3 low expression. Thus, these findings indicated that BCL3 appeared as a promising molecular biomarker of pediatric acute myeloid leukemia with unfavorable prognosis.

Dual-Mode Dark Field and Surface-Enhanced Raman Scattering Liposomes for Lymphoma and Leukemia Cell Imaging.

Multifunctional probes are needed to characterize individual cells simultaneously by different techniques to provide complementary information. A preparative method and an in vitro demonstration of function are presented for a dual-function dark field microscopy/surface-enhanced Raman scattering (SERS) liposome probe for cancer. Liposomes composed of zwitterionic lipids are valuable both to limit biofouling and to serve as a modular matrix to incorporate a variety of functional molecules and hence are used here as vehicles for SERS-active materials. Dark field microscopy and SERS represent new combined functionalities for targeted liposomal probes. Two methods of antibody conjugation to SERS liposomes are demonstrated: (i) direct conjugation to functional groups on the SERS liposome surface and (ii) postinsertion of lipid-functionalized antibody fragments (Fabs) into preformed SERS liposomes. In vitro experiments targeting both lymphoma cell line LY10 and primary human chronic lymphocytic leukemia (CLL) cells demonstrate the usefulness of these probes as optical contrast agents in both dark field and Raman microscopy.

Splicing modulation sensitizes chronic lymphocytic leukemia cells to venetoclax by remodeling mitochondrial apoptotic dependencies.

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

T Cell Leukemia/Lymphoma 1A is essential for mouse epidermal keratinocytes proliferation promoted by insulin-like growth factor 1.

T Cell Leukemia/Lymphoma 1A is expressed during B-cell differentiation and, when over-expressed, acts as an oncogene in mouse (Tcl1a) and human (TCL1A) B-cell chronic lymphocytic leukemia (B-CLL) and T-cell prolymphocytic leukemia (T-PLL). Furthermore, in the murine system Tcl1a is expressed in the ovary, testis and in pre-implantation embryos, where it plays an important role in blastomere proliferation and in embryonic stem cell (ESC) proliferation and self-renewal. We have also observed that Tcl1-/- adult mice exhibit alopecia and deep ulcerations. This finding has led us to investigate the role of TCL1 in mouse skin and hair follicles. We have found that TCL1 is expressed in the proliferative structure (i.e. the secondary hair germ) and in the stem cell niche (i.e. the bulge) of the hair follicle during regeneration phase and it is constitutively expressed in the basal layer of epidermis where it is required for the correct proliferative–differentiation program of the keratinocytes (KCs). Taking advantage of the murine models we have generated, including the Tcl1-/- and the K14-TCL1 transgenic mouse, we have analysed the function of TCL1 in mouse KCs and the molecular pathways involved. We provide evidence that in the epidermal compartment TCL1 has a role in the regulation of KC proliferation, differentiation, and apoptosis. In particular, the colony-forming efficiency (CFE) and the insulin-like growth factor 1 (IGF1)-induced proliferation are dramatically impaired, while apoptosis is increased, in KCs from Tcl1-/- mice when compared to WT. Moreover, the expression of differentiation markers such as cytokeratin 6 (KRT6), filaggrin (FLG) and involucrin (IVL) are profoundly altered in mutant mice (Tcl1-/-). Importantly, by over-expressing TCL1A in basal KCs of the K14-TCL1 transgenic mouse model, we observed a significant rescue of cell proliferation, differentiation and apoptosis of the mutant phenotype. Finally, we found TCL1 to act, at least in part, via increasing phospho-ERK1/2 and decreasing phospho-P38 MAPK. Hence, our data demonstrate that regulated levels of Tcl1a are necessary for the correct proliferation and differentiation of the interfollicular KCs.

Exome sequencing of the TCL1 mouse model for CLL reveals genetic heterogeneity and dynamics during disease development

The TCL1 mouse model is widely used to study pathophysiology, clonal evolution, and drug sensitivity or resistance of chronic lymphocytic leukemia (CLL). By performing whole exome sequencing, we present the genetic landscape of primary tumors from TCL1 mice and of TCL1 tumors serially transplanted into wild-type recipients to mimic clonal evolution. We show that similar to CLL patients, mutations in mice are frequently subclonal and heterogenous among different primary TCL1 mice. We further describe that this molecular heterogeneity mirrors heterogenous disease characteristics such as organ infiltration or CLL dependent T cell skewing. Similar to human CLL, we further observed the occurrence of novel mutations and structural variations during clonal evolution and found plasticity in the expansion of B cell receptor specific subclones. Thus, our results uncover that the genetic complexity, pathway dependence and clonal dynamics in mouse CLL are in relevant agreement to human CLL, and they are important to consider in future research using the TCL1 mouse for studying CLL.

Identification of Distinct Unmutated Chronic Lymphocytic Leukemia Subsets in Mice Based on Their T Cell Dependency

Chronic lymphocytic leukemia (CLL) can be divided into prognostically distinct subsets with stereotyped or non-stereotyped, mutated or unmutated B cell receptors (BCRs). Individual subsets vary in antigen specificity and origin, but the impact of antigenic pressure on the CLL BCR repertoire remains unknown. Here, we employed IgH.TEμ mice that spontaneously develop CLL, expressing mostly unmutated BCRs of which ~35% harbor VH11-2/Vκ14-126 and recognize phosphatidylcholine. Proportions of VH11/Vκ14-expressing CLL were increased in the absence of functional germinal centers in IgH.TEμ mice deficient for CD40L or activation-induced cytidine deaminase. Conversely, in vivo T cell-dependent immunization decreased the proportions of VH11/Vκ14-expressing CLL. Furthermore, CLL onset was accelerated by enhanced BCR signaling in Siglec-G−/− mice or in mice expressing constitutively active Bruton's tyrosine kinase. Transcriptional profiling revealed that VH11 and non-VH11 CLL differed in the upregulation of specific pathways implicated in cell signaling and metabolism. Interestingly, principal component analyses using the 148 differentially expressed genes revealed that VH11 and non-VH11 CLL clustered with BCR-stimulated and anti-CD40-stimulated B cells, respectively. We identified an expression signature consisting of 13 genes that were differentially expressed in a larger panel of T cell-dependent non-VH11 CLL compared with T cell-independent VH11/Vκ14 or mutated IgH.TEμ CLL. Parallel differences in the expression of these 13 signature genes were observed between heterogeneous and stereotypic human unmutated CLL. Our findings provide evidence for two distinct unmutated CLL subsets with a specific transcriptional signature: one is T cell-independent and B-1 cell-derived while the other arises upon antigen stimulation in the context of T-cell help.

Fludarabine-resistance associates with ceramide metabolism and leukemia stem cell development in chronic lymphocytic leukemia.

Fludarabine (flu) -containing regimens such as flu, cyclophosphamide and rituximab have been established as one of the standard first line therapy in medically-fit chronic lymphocytic leukemia (CLL) patients. Therefore, flu-refractory (primary flu-insensitivity or flu-caused relapse) remains a major problem causing treatment failure for CLL patients. We isolated the peripheral blood mononuclear cells (PBMCs) from CLL patients and treated with flu to find flu-refractory cases, and established flu-resistant clonal cells to study molecular mechanism of flu-resistance. By comparing parental MEC-2 cells, a human CLL cell line, we found that flu-resistant clonal cells were significantly increased lethal dose 50 of flu concentration, and up-regulated expression of P-glycoprotein, a drug-resistant marker, glucosylceramide synthase (GCS), an enzyme that can convert ceramide to glucosylceramide, and CD34, a leukemia stem cell marker. Overexpression of GCS leads to promptly elimination of cellular ceramide levels and accumulation of glucosylceramide, which reduces apoptosis and promotes survival and proliferation of flu-resistant clonal cells. Furthermore, we demonstrated that the accumulation of glucosylceramide can be blocked by PDMP to restore flu-sensitivity in flu-resistant clonal cells. We also found that elevating glucosylceramide levels in flu-resistant clonal cells was associated with up-regulation of GCS and CD34 expression. Importantly, overexpression of GCS or CD34 was also determined in flu-refractory PBMCs. Our results show that flu-resistance is associated with the alteration of ceramide metabolism and the development of leukemia stem cell-like cells. The flu-resistance can be reversed by GCS inhibition as a novel strategy for overcoming drug resistance.

Abstract 1870: The anti-proliferative and pro-apoptotic effect of MDM2-p53 antagonists evaluated in human tumor cells lines and chronic lymphocytic leukemia patient samples

Abstract We investigated the cellular response to two MDM2-p53 antagonists (RG7388 and Cpd 1) in a panel of 19 tumor cell lines and in patient-derived chronic lymphocytic leukemia (CLL) cells. RG7388 (Idasanutlin) is an MDM2-p53 antagonist developed by Roche and Cpd1 a novel potent and selective MDM2-p53 antagonist developed as part of an ongoing Alliance between Newcastle University, Cancer Research UK and Astex Pharmaceuticals. RG7388 and Cpd1 potently inhibited the proliferation of many cell lines, with MDM2-amplified SJSA-1 osteosarcoma cells and cells from haematological malignancies being the most sensitive (e.g. GI50 values in the acute myeloid leukemia cell line MOLM13 were 33 + 16 nM for RG7388 and 8 + 1 nM for Cpd 1). We also examined induction of apoptosis by measuring caspase 3/7 activation (24 h treatment). SJSA-1 cells showed a 20-fold increase in caspase activation and Molm13 cells a 6-fold increase (measured at 300nM with RG7388). However, cell lines from solid tumours such as the colorectal carcinoma HCT116 or the hepatocellular carcinoma HepG2 did not show any induction of apoptosis, even at concentrations up to 1µM RG3788. In primary CLL cells, Cpd 1 reduced cell viability (48 h) with an average LC50 of 131 + 46 nM (n = 4), compared to 400 + 55 nM for RG7388 (n =23). We also quantified mRNA levels in 25 primary CLL samples and found that RG7388 induced a predominantly pro-apoptotic gene signature: following 6h treatment, 1µM RG7388 resulted in an average 8-fold induction of PUMA and 3.5-fold increase in BAX compared to a 1.6-fold induction of CDKN1A (p21). Our data confirm that targeting the MDM2-p53 interaction is an effective strategy to inhibit growth and viability of tumor cells, and that some cells, including those expressing high levels of MDM2 or those derived from hematological malignancies, display a striking apoptotic response. Citation Format: Elaine Willmore, Yan Zhao, Carmela Ciardullo, Laura Woodhouse, Huw D. Thomas, Maria Ahn, Lynsey Fazal, Martin E. Noble, Ian Hardcastle, Steven Howard, Gianni Chessari, John Lunec, Steve Wedge. The anti-proliferative and pro-apoptotic effect of MDM2-p53 antagonists evaluated in human tumor cells lines and chronic lymphocytic leukemia patient samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1870.

Abstract 4756: Secretory IgM exacerbates tumor progression by inducing accumulations of myeloid-derived suppressor cells in mice

Abstract To explore the role of B cell receptor (BCR) in promoting malignant progression of chronic lymphocytic leukemia (CLL) in mice, we generate MD4/Eμ-TCL1 mice, whose B cells harbor a monoclonal BCR against hen egg lysozyme (HEL) and secrete IgM against HEL. MD4/Eμ-TCL1 mice survive significantly shorter than Eμ-TCL1 mice. While precancerous B cells in young MD4/Eμ-TCL1 mice recognize HEL, CLL cells developed in older MD4/Eμ-TCL1 mice fail to recognize HEL. Nevertheless, MD4/Eμ-TCL1 CLL cells can be activated by goat F(ab')2 anti-mouse IgM and respond by phosphorylation of Igα, Syk and ERK1/2, indicating reactivation of a parental Ig gene allele. MD4/Eμ-TCL1 CLL cells also produce large quantities of secretory IgM (sIgM), which does not react with HEL. Compared with age-matched Eμ-TCL1 mice, MD4/Eμ-TCL1 mice also generate a significantly increased population of CD11b+/Ly6G+ granulocytic cells in the peripheral blood, spleens and bone marrow. CD11b+/Ly6G+ granulocytic cells purified from spleens of MD4/Eμ-TCL1 mice can suppress CD3/CD28-mediated proliferation of CD8+ T cells and gp100-loaded class I MHC-mediated proliferation of CD8+ T cells from PMEL-1 mice, qualifying these cells as myeloid-derived suppressor cells (MDSCs). Increased MDSCs may account for significantly decreased T cells and poor prognosis in CLL-bearing MD4/Eμ-TCL1 mice. Because MD4/Eμ-TCL1 mouse CLL cells and human CLL cells can produce sIgM, we hypothesize that sIgM may account for the accumulation of MDSCs. To test this hypothesis, we cross μS-/- mice, which cannot produce sIgM, with Eμ-TCL1 mice. The μS-/-/Eμ-TCL1 mice indeed develop significantly lower numbers of MDSCs, and survive significantly longer than Eμ-TCL1 mice. We decide to target the synthesis of sIgM by deleting the function of XBP-1, because the synthesis of sIgM is tightly regulated by a mechanism called regulated IRE-1-dependent decay (RIDD), which is hyperactivated in B cells as a response to XBP-1 deficiency. We cross B cell-specific XBP-1KO mice with MD4/Eμ-TCL1 mice, and the resultant XBP-1KO/MD4/Eμ-TCL1 mice indeed produce significantly reduced amounts of sIgM and decreased numbers of MDSCs. In addition, μS-/- mice grafted with Lewis lung carcinoma exhibit reduced functions of MDSCs in suppressing T cells, resulting in significantly slower tumor growth. These results clearly demonstrate that sIgM produced by B cells can upregulate the immunosuppressive functions of MDSCs in tumor-bearing mice to aggravate cancer progression. Citation Format: Chih-Hang Tang, Shiun Chang, Ayumi Hashimoto, Yi-Ju Chen, Chang Won Kang, Anthony Mato, Juan Del Valle, Dmitry Gabrilovich, Chih-Chi Andrew Hu. Secretory IgM exacerbates tumor progression by inducing accumulations of myeloid-derived suppressor cells in mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4756.

Potent anti-leukemic activity of a specific cyclin-dependent kinase 9 inhibitor in mouse models of chronic lymphocytic leukemia.

Onset of progression even during therapy with novel drugs remains an issue in chronic lymphocytic leukemia (CLL). Thus, there is ongoing demand for novel agents. Approaches targeting cyclin-dependent kinases (CDK) have reached the clinical trial stage. CDK9 mediating RNA transcriptional elongation is the evolving pivotal CLL CDK inhibitor target. However, more CDK9 selective compounds are desirable. Here, we describe the CDK9 inhibitor LDC526 displaying a low nanomolar biochemical activity against CDK9 and an at least 50-fold selectivity against other CDKs. After demonstrating in vitro MEC-1 cell line and primary human CLL cell cytotoxicity we evaluated the LDC526 in vivo effect on human CLL cells transplanted into NOD/scid/γcnull (NSG) mice. LDC526 administration (75 mg/kg) for 5 days resulted in a 77% reduction of human CLL cells in NSG spleens compared to carrier control treatment. Next, we longitudinally studied the LDC526 impact on circulating CLL cells in the TCL1 transgenic mouse model. LDC526 (50 mg/kg) administration for two days led to a 16-fold reduction of blood CLL cell numbers. Remarkably, residual CLL cells exhibited significantly increased intracellular BCL-2 levels. However, the LDC526 cytotoxic effect was not restricted to CLL cells as also declining numbers of normal B and T lymphocytes were observed in LDC526 treated TCL1 mice. Taken together, our in vivo data provide a strong rational for continued LDC526 development in CLL therapy and argue for the combination with BCL-2 inhibitors.

GPCRomics: GPCR Expression in Cancer Cells and Tumors Identifies New, Potential Biomarkers and Therapeutic Targets.

G protein-coupled receptors (GPCRs), the largest family of targets for approved drugs, are rarely targeted for cancer treatment, except for certain endocrine and hormone-responsive tumors. Limited knowledge regarding GPCR expression in cancer cells likely has contributed to this lack of use of GPCR-targeted drugs as cancer therapeutics. We thus undertook GPCRomic studies to define the expression of endoGPCRs (which respond to endogenous molecules such as hormones, neurotransmitters and metabolites) in multiple types of cancer cells. Using TaqMan qPCR arrays to quantify the mRNA expression of ∼340 such GPCRs, we found that human chronic lymphocytic leukemia (CLL) cells/stromal cells associated with CLL, breast cancer cell lines, colon cancer cell lines, pancreatic ductal adenocarcinoma (PDAC) cells, cancer associated fibroblasts (CAFs), and PDAC tumors express 50 to >100 GPCRs, including many orphan GPCRs (which lack known physiologic agonists). Limited prior data exist regarding the expression or function of most of the highly expressed GPCRs in these cancer cells and tumors. Independent results from public cancer gene expression databases confirm the expression of such GPCRs. We propose that highly expressed GPCRs in cancer cells (for example, GPRC5A in PDAC and colon cancer cells and GPR68 in PDAC CAFs) may contribute to the malignant phenotype, serve as biomarkers and/or may be novel therapeutic targets for the treatment of cancer.

B-cell tumor development in Tet2-deficient mice

AbstractThe TET2 gene encodes an α-ketoglutarate–dependent dioxygenase able to oxidize 5-methylcytosine into 5-hydroxymethylcytosine, which is a step toward active DNA demethylation. TET2 is frequently mutated in myeloid malignancies but also in B- and T-cell malignancies. TET2 somatic mutations are also identified in healthy elderly individuals with clonal hematopoiesis. Tet2-deficient mouse models showed widespread hematological differentiation abnormalities, including myeloid, T-cell, and B-cell malignancies. We show here that, similar to what is observed with constitutive Tet2-deficient mice, B-cell–specific Tet2 knockout leads to abnormalities in the B1-cell subset and a development of B-cell malignancies after long latency. Aging Tet2-deficient mice accumulate clonal CD19+ B220low immunoglobulin M+ B-cell populations with transplantable ability showing similarities to human chronic lymphocytic leukemia, including CD5 expression and sensitivity to ibrutinib-mediated B-cell receptor (BCR) signaling inhibition. Exome sequencing of Tet2−/− malignant B cells reveals C-to-T and G-to-A mutations that lie within single-stranded DNA–specific activation-induced deaminase (AID)/APOBEC (apolipoprotein B messenger RNA editing enzyme, catalytic polypeptide-like) cytidine deaminases targeted motif, as confirmed by the lack of a B-cell tumor in compound Tet2-Aicda–deficient mice. Finally, we show that Tet2 deficiency accelerates and exacerbates T-cell leukemia/lymphoma 1A–induced leukemogenesis. Together, our data establish that Tet2 deficiency predisposes to mature B-cell malignancies, which development might be attributed in part to AID-mediated accumulating mutations and BCR-mediated signaling.

Casein kinase 1 is a therapeutic target in chronic lymphocytic leukemia

AbstractCasein kinase 1δ/ε (CK1δ/ε) is a key component of noncanonical Wnt signaling pathways, which were shown previously to drive pathogenesis of chronic lymphocytic leukemia (CLL). In this study, we investigated thoroughly the effects of CK1δ/ε inhibition on the primary CLL cells and analyzed the therapeutic potential in vivo using 2 murine model systems based on the Eµ-TCL1–induced leukemia (syngeneic adoptive transfer model and spontaneous disease development), which resembles closely human CLL. We can demonstrate that the CK1δ/ε inhibitor PF-670462 significantly blocks microenvironmental interactions (chemotaxis, invasion and communication with stromal cells) in primary CLL cells in all major subtypes of CLL. In the mouse models, CK1 inhibition slows down accumulation of leukemic cells in the peripheral blood and spleen and prevents onset of anemia. As a consequence, PF-670462 treatment results in a significantly longer overall survival. Importantly, CK1 inhibition has synergistic effects to the B-cell receptor (BCR) inhibitors such as ibrutinib in vitro and significantly improves ibrutinib effects in vivo. Mice treated with a combination of PF-670462 and ibrutinib show the slowest progression of disease and survive significantly longer compared with ibrutinib-only treatment when the therapy is discontinued. In summary, this preclinical testing of CK1δ/ε inhibitor PF-670462 demonstrates that CK1 may serve as a novel therapeutic target in CLL, acting in synergy with BCR inhibitors. Our work provides evidence that targeting CK1 can represent an alternative or addition to the therapeutic strategies based on BCR signaling and antiapoptotic signaling (BCL-2) inhibition.

Preferential use of unmutated immunoglobulin heavy variable region genes in Boxer dogs with chronic lymphocytic leukemia.

Human chronic lymphocytic leukemia (CLL) is a clinically heterogeneous disease, and immunoglobulin heavy variable region (IGHV) gene mutational status is an important prognostic marker. IGHV mutational status has not been previously examined in canine CLL. We sequenced the IGHV-D-J rearrangements from 55 canine patients with CLL, including 36 non-Boxer and 19 Boxer dogs. The majority of non-Boxers (75%) had mutated IGHV genes, whereas the majority of Boxers (79%) had unmutated IGHV genes. IGHV3-41 and IGHV3-67 gene usage was significantly higher in Boxers with CLL compared to non-Boxers. Additionally, 11 Boxers with large B-cell lymphoma and the normal IGHV repertoire of six control dogs (three Boxers and three non-Boxers) were sequenced. IGHV3-41 was preferentially used in Boxers with other forms of lymphoma and without lymphoproliferative disease. However, preferential use of unmutated IGHV genes was unique to Boxers with CLL, suggesting Boxers may be a valuable model to investigate unmutated CLL.

Abstract A115: Targeting glycolysis and RAD51-dependent repair in B-lymphoid cancers

Abstract Activation-Induced Cytidine Deaminase (AID) mutates and modifies the IgH locus. However, it also initiates double-strand breaks (DSBs) throughout the genome. This effect of AID is balanced by RAD51-dependent homologous recombination repair. Importantly, we previously showed that inhibition of RAD51 function by a small-molecule inhibitor leads to cell death of AID-positive neoplastic cells. While we have used AID-RAD51 imbalance as a potential chemotherapy approach, the efficacy of current RAD51 inhibitors can be further improved. Studies have shown a relationship between glycolysis and RAD51 function, and therefore targeting the glycolytic pathway is a viable strategy to enhance RAD51 inhibitor efficacy. Here we show that glycolytic inhibitor 2DG and RAD51 inhibitor DIDS act synergistically to reduce B-lymphocyte human chronic lymphocytic leukemia proliferation ex vivo, and mice xenografted with this cell line showed significant reduction in the tumor burden in the presence of both 2DG and DIDS. These data suggest that the glycolytic pathway and DNA repair can be simultaneously targeted to produce better chemotherapies against B-lymphoid cancers. Citation Format: Muneer G. Hasham, Kin-hoe Chow, John Wilson, Nathan LaBrie, Bryant Perkins, Jane Branca, Thomas Sproule, Derry Roopenian. Targeting glycolysis and RAD51-dependent repair in B-lymphoid cancers [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A115.

Two mouse models reveal an actionable PARP1 dependence in aggressive chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) remains an incurable disease. Two recurrent cytogenetic aberrations, namely del(17p), affecting TP53, and del(11q), affecting ATM, are associated with resistance against genotoxic chemotherapy (del17p) and poor outcome (del11q and del17p). Both del(17p) and del(11q) are also associated with inferior outcome to the novel targeted agents, such as the BTK inhibitor ibrutinib. Thus, even in the era of targeted therapies, CLL with alterations in the ATM/p53 pathway remains a clinical challenge. Here we generated two mouse models of Atm- and Trp53-deficient CLL. These animals display a significantly earlier disease onset and reduced overall survival, compared to controls. We employed these models in conjunction with transcriptome analyses following cyclophosphamide treatment to reveal that Atm deficiency is associated with an exquisite and genotype-specific sensitivity against PARP inhibition. Thus, we generate two aggressive CLL models and provide a preclinical rational for the use of PARP inhibitors in ATM-affected human CLL.

Abstract 2449: Transcriptome and whole-genome sequencing analysis of a novel murine model of chronic lymphocytic leukemia

Abstract Large-scale cancer sequencing of primary chronic lymphocytic leukemia (CLL) has identified SF3B1, an RNA splicing factor, as one of the most frequently mutated CLL genes. SF3B1 mutations localize to a hotspot (>50% at K700E site) and highly co-occur with mutations in ATM or deletion of chromosome 11q (minimally deleted region contains ATM). How this splicing factor mutation alone or in cooperation with ATM deletion contributes to CLL remains elusive. Genetically engineered mice are powerful tools in understanding genetic lesions and cancer phenotypes. We therefore generated a mouse line that conditionally expresses heterozygous Sf3b1-K700E mutation. We modeled the effects of the combined alterations by crossing mice with conditional knockout of Atm and mice with Sf3b1-K700E. By breeding these mice with homozygous CD19-Cre transgenic mice, we achieved B cell-restricted expression of heterozygous Sf3b1 mutation and Atm deletion. B cell co-expression of these two mutations in vivo led to clonal expansion of CD19+CD5+ B cells in blood, marrow and spleen in aged mice (18 to 24-month old) at low penetrance. No leukemia cells were found in the Sf3b1-K700E mice (up to 24-month old). The CLL cells from the double mutant mice could be engrafted in both immunocompetent and immunodeficient mice, with detectable disease within 2-4 weeks following transfer, thus making this mouse line amenable to drug discovery and biologic investigations. To investigate how Sf3b1 mutation and Atm deletion synergistically contribute to CLL, we asked if there are RNA and DNA level changes in the double mutant mice with CLL. First, we performed transcriptome sequencing of splenic B cell RNA collected from age-matched mice that either express wild-type, singly mutant alleles of Sf3b1 or Atm, or doubly mutant alleles with or without CLL-like disease. Using the tool JuncBASE, we classified and quantified splice variants associated with the different genetic alterations. Consistent with prior findings in human CLL, we observed that splice variants in Sf3b1-mutant mice alone were highly enriched at 3' splice sites. Sf3b1 and Atm doubly mutant B cells displayed a splicing pattern similar to that in Sf3b1 singly mutant cells. Moreover, we identified unique CLL splice variants in genes (Setdb2, Phf11c) previously shown to be associated with CLL. Next, we examined the mutation rate in DNA derived from splenic B cells collected from mice with a singly mutated allele of Sf3b1 or Atm, or with doubly mutated alleles with and without CLL-like disease through comparison against matched germline DNA from kidney by whole-genome sequencing. We have observed that co-expression of Sf3b1 mutation and deletion of Atm results in a higher mutation rate compared to singly mutant cells. Our analysis has revealed that altered RNA splicing and genomic instability all contribute to CLL leukemogenesis. We will further dissect how the two lesions contribute to CLL functionally using this model. Citation Format: Lili Wang, Jing Sun, Amaro Taylor-Weiner, Jaegil Kim, Zachary J. Cartun, Angela N. Brooks, Donna Neuberg, Mark D. Fleming, Benjamin L. Ebert, Gad Getz, Ruben Carrasco, Catherine J. Wu. Transcriptome and whole-genome sequencing analysis of a novel murine model of chronic lymphocytic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2449. doi:10.1158/1538-7445.AM2017-2449

The Bruton Tyrosine Kinase (BTK) Inhibitor Acalabrutinib Demonstrates Potent On-Target Effects and Efficacy in Two Mouse Models of Chronic Lymphocytic Leukemia.

Purpose: Acalabrutinib (ACP-196) is a novel, potent, and highly selective Bruton tyrosine kinase (BTK) inhibitor, which binds covalently to Cys481 in the ATP-binding pocket of BTK. We sought to evaluate the antitumor effects of acalabrutinib treatment in two established mouse models of chronic lymphocytic leukemia (CLL).Experimental Design: Two distinct mouse models were used, the TCL1 adoptive transfer model where leukemic cells from Eμ-TCL1 transgenic mice are transplanted into C57BL/6 mice, and the human NSG primary CLL xenograft model. Mice received either vehicle or acalabrutinib formulated into the drinking water.Results: Utilizing biochemical assays, we demonstrate that acalabrutinib is a highly selective BTK inhibitor as compared with ibrutinib. In the human CLL NSG xenograft model, treatment with acalabrutinib demonstrated on-target effects, including decreased phosphorylation of PLCγ2, ERK, and significant inhibition of CLL cell proliferation. Furthermore, tumor burden in the spleen of the mice treated with acalabrutinib was significantly decreased compared with vehicle-treated mice. Similarly, in the TCL1 adoptive transfer model, decreased phosphorylation of BTK, PLCγ2, and S6 was observed. Most notably, treatment with acalabrutinib resulted in a significant increase in survival compared with mice receiving vehicle.Conclusions: Treatment with acalabrutinib potently inhibits BTK in vivo, leading to on-target decreases in the activation of key signaling molecules (including BTK, PLCγ2, S6, and ERK). In two complementary mouse models of CLL, acalabrutinib significantly reduced tumor burden and increased survival compared with vehicle treatment. Overall, acalabrutinib showed increased BTK selectivity compared with ibrutinib while demonstrating significant antitumor efficacy in vivo on par with ibrutinib. Clin Cancer Res; 23(11); 2831-41. ©2016 AACR.

Effective control of acute myeloid leukaemia and acute lymphoblastic leukaemia progression by telomerase specific adoptive T-cell therapy.

Telomerase (TERT) is a ribonucleoprotein enzyme that preserves the molecular organization at the ends of eukaryotic chromosomes. Since TERT deregulation is a common step in leukaemia, treatments targeting telomerase might be useful for the therapy of hematologic malignancies. Despite a large spectrum of potential drugs, their bench-to-bedside translation is quite limited, with only a therapeutic vaccine in the clinic and a telomerase inhibitor at late stage of preclinical validation. We recently demonstrated that the adoptive transfer of T cell transduced with an HLA-A2-restricted T-cell receptor (TCR), which recognize human TERT with high avidity, controls human B-cell chronic lymphocytic leukaemia (B-CLL) progression without severe side-effects in humanized mice. In the present report, we show the ability of our approach to limit the progression of more aggressive leukemic pathologies, such as acute myeloid leukaemia (AML) and B-cell acute lymphoblastic leukaemia (B-ALL). Together, our findings demonstrate that TERT-based adoptive cell therapy is a concrete platform of T cell-mediated immunotherapy for leukaemia treatment.

APOBEC gene family expression and hallmarks in chronic lymphocytic leukemia

Abstract The hallmark activity of APOBEC (apolipoprotein B mRNA editing enzyme, catalytic polypeptide) family of cytidine deaminases, including activation-induced deaminase (AID) and APOBEC3 genes, has been detected in somatic mutation signatures by ultra-deep sequencing of the genomes of many cancers, including chronic lymphocytic leukemia (CLL). The acquisition of these mutations is hypothesized to lead to the progression towards aggressive disease in cancer. To examine this in CLL, we tested if increased APOBEC family member gene expression in CLL cells, as measured by microarray and quantitative real time PCR, correlated with worse patient outcome. Higher levels of AID, APOBEC3B, APOBEC3F and APOBEC3H in CLL cells correlated with worse patient outcome, whereas APOBEC3G did not. Interestingly, higher levels of a truncated splice variant of APOBEC3F tended to correlate with better patient outcome. The expression of truncated APOBEC3F may possibly interfere with APOBEC family member mutation activity. To test mutation activity, CLL cells were activated by transfer into NOD-scid IL2Rγnull mice, a xenograft model of human CLL, and hallmark mutation signatures in the expressed immunoglobulin variable region (IGHV) of CLL cells were analyzed by targeted ultra-deep sequencing. Induced IGHV mutation hallmarks consistent with AID were found. These data support the hypothesis that expression and mutation activity of APOBEC family members, such as AID, in CLL cells could lead to adverse patient consequences.