Profiling the activity of the para-caspase MALT1 in B-cell acute lymphoblastic leukemia for potential targeted therapeutic application.

B cell acute lymphoblastic leukemia (B-ALL) remains a hard-to-treat disease with a poor prognosis in adults. Mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) is a para-caspase required for B-cell receptor (BCR)-mediated NF-κB activation. Inhibition of MALT1 in preclinical models has proven efficacious in many B-cell malignancies including chronic lymphocytic leukemia, mantle cell lymphoma and diffuse large B-cell lymphoma. We sought to examine the role of MALT1 in B-ALL and determine the biological consequences of its inhibition. Targeting MALT1 with both Z-VRPR-fmk and MI-2 efficiently kills B-ALL cells independent of the cell-of-origin (pro, pre, mature) or the presence of the Philadelphia chromosome, and spares normal B-cells. The mechanism of cell death was through apoptotic induction, mostly in cycling cells. The proteolytic activity of MALT1 can be studied by measuring its ability to cleave its substrates. Surprisingly, with the exception of mature B-ALL, we did not detect cleavage of MALT1 substrates at baseline, nor after proteasomal inhibition or following activation of pre-BCR. To explore the possibility of a distinct role for MALT1 in B-ALL, independent of signaling through BCR, we studied the changes in gene expression profiling following a 24-hour treatment with MI-2 in 12 B-ALL cell lines. Our transcriptome analysis revealed a strong inhibitory effect on MYC-regulated gene signatures, further confirmed by Myc protein downregulation, concomitant with an increase in the Myc degrader FBXW7. In conclusion, our evidence suggests a novel role for MALT1 in B-ALL through Myc regulation and provides support for clinical testing of MALT1 inhibitors in B-ALL.

Insulin-Like Growth Factor-1 Receptor Deficiency in Macrophages Accelerates Atherosclerosis and Induces an Unstable Plaque Phenotype in Apolipoprotein E-Deficient Mice.

BACKGROUND: We have previously shown that systemic infusion of insulin-like growth factor-1 (IGF-1) exerts anti-inflammatory and antioxidant effects and reduces atherosclerotic burden in apolipoprotein E (Apoe)-deficient mice. Monocytes/macrophages express high levels of IGF-1 receptor (IGF1R) and play a pivotal role in atherogenesis, but the potential effects of IGF-1 on their function are unknown. METHODS AND RESULTS: To determine mechanisms whereby IGF-1 reduces atherosclerosis and to explore the potential involvement of monocytes/macrophages, we created monocyte/macrophage-specific IGF1R knockout (MΦ-IGF1R-KO) mice on an Apoe(-/-) background. We assessed atherosclerotic burden, plaque features of stability, and monocyte recruitment to atherosclerotic lesions. Phenotypic changes of IGF1R-deficient macrophages were investigated in culture. MΦ-IGF1R-KO significantly increased atherosclerotic lesion formation, as assessed by Oil Red O staining of en face aortas and aortic root cross-sections, and changed plaque composition to a less stable phenotype, characterized by increased macrophage and decreased α-smooth muscle actin-positive cell population, fibrous cap thinning, and decreased collagen content. Brachiocephalic artery lesions of MΦ-IGF1R-KO mice had histological features implying plaque vulnerability. Macrophages isolated from MΦ-IGF1R-KO mice showed enhanced proinflammatory responses on stimulation by interferon-γ and oxidized low-density lipoprotein and elevated antioxidant gene expression levels. Moreover, IGF1R-deficient macrophages had decreased expression of ABCA1 and ABCG1 and reduced lipid efflux. CONCLUSIONS: Our data indicate that macrophage IGF1R signaling suppresses macrophage and foam cell accumulation in lesions and reduces plaque vulnerability, providing a novel mechanism whereby IGF-1 exerts antiatherogenic effects.

Reduction in Cell Viability and in Homeobox Protein Levels Following in Vitro Exposure to δ-tocopherol in Acute Myeloid Leukemia.

δ-Tocopherol (δ-T), the least prevalent tocopherol in our diet, was described to have a more potent anticancer activity in solid tumors compared to the other tocopherols. δ-T induces tumor cell death through peroxisome proliferator-activated receptor γ (PPAR-γ) induction, cyclin-D1 inhibition, and modulation of redox balance. Nevertheless, the role of δ-T in preventing or treating hematologic malignancies has not been studied. In this study, we screened the efficacy of δ-T against six cell lines representing a wide spectrum of hematologic malignancies: Jurkat (acute T-cell leukemia), K-562 (chronic myeloid leukemia), KG-1 [acute myeloid leukemia (AML)], THP-1 (acute monocytic leukemia), TOM-1 (acute lymphoblastic leukemia), and UMCL01-101 (AIDS-associated diffuse large B-cell lymphoma). Interestingly, the AML cell line KG-1 was the only one to be significantly affected at concentrations of δ-T as low as 20 µM. The antileukemic activity of δ-T in AML was verified in a set of primary cells collected from patients newly diagnosed with AML. Apoptotic induction and cell cycle arrest explained the efficacy of δ-T against KG-1 cells. The mechanism of cell growth inhibition of δ-T was through downregulation of cyclin-D1 and a set of homeobox proteins (HOXA9, PBX1, and Cdx2) that have a well-documented role in the pathobiology of AML.

The surface glycoprotein of feline leukemia virus isolate FeLV-945 is a determinant of altered pathogenesis in the presence or absence of the unique viral long terminal repeat.

Feline leukemia virus (FeLV) is a naturally transmitted gammaretrovirus that infects domestic cats. FeLV-945, the predominant isolate associated with non-T-cell disease in a natural cohort, is a member of FeLV subgroup A but differs in sequence from the FeLV-A prototype, FeLV-A/61E, in the surface glycoprotein (SU) and long terminal repeat (LTR). Substitution of the FeLV-945 LTR into FeLV-A/61E resulted in pathogenesis indistinguishable from that of FeLV-A/61E, namely, thymic lymphoma of T-cell origin. In contrast, substitution of both FeLV-945 LTR and SU into FeLV-A/61E resulted in multicentric lymphoma of non-T-cell origin. These results implicated the FeLV-945 SU as a determinant of pathogenic spectrum. The present study was undertaken to test the hypothesis that FeLV-945 SU can act in the absence of other unique sequence elements of FeLV-945 to determine the disease spectrum. Substitution of FeLV-A/61E SU with that of FeLV-945 altered the clinical presentation and resulted in tumors that demonstrated expression of CD45R in the presence or absence of CD3. Despite the evident expression of CD45R, a typical B-cell marker, T-cell receptor beta (TCRß) gene rearrangement indicated a T-cell origin. Tumor cells were detectable in bone marrow and blood at earlier times during the disease process, and the predominant SU genes from proviruses integrated in tumor DNA carried markers of genetic recombination. The findings demonstrate that FeLV-945 SU alters pathogenesis, although incompletely, in the absence of FeLV-945 LTR. Evidence demonstrates that FeLV-945 SU and LTR are required together to fully recapitulate the distinctive non-T-cell disease outcome seen in the natural cohort.

Establishment and characterization of a new mantle cell lymphoma cell line with a NOTCH2 mutation, Arbo.

Cell lines represent an essential tool used in preclinical research. Most hematologic malignancies have a wide array of cell lines representing their respective molecular and pathologic spectra. In mantle cell lymphoma (MCL), cell lines become specifically valuable in view of the heterogeneity of this disease. Unfortunately, the number of MCL cell lines that are available for the research community remains small, with only nine cell lines available for purchase through the American Type Culture Collection (ATCC). We have established a novel blastoid MCL cell line, isolated from the malignant pleural effusion of a 69-year-old male with refractory MCL. Arbo was fully characterized with cytogenetics, immunophenotyping, whole exome sequencing and drug sensitivity assays. One of the most notable mutations identified in Arbo (but not in normal tissue) was the missense mutation NOTCH2 R2400*, which has been proposed as a clinically significant mutation in MCL seen in 5% of cases. NOTCH2 R2400* results in a truncated Notch2 protein, leading to a more stable and active protein. Using pharmacologic inhibition of Notch2, we showed a dependence of Arbo on NOTCH2 signaling, as well as a link between CD23 expression on Arbo and NOTCH2 activity. Arbo represents a NOTCH2 mutated model that is useful in MCL as well as other lymphomas with such mutation. We plan to deposit Arbo at the ATCC to be available for the research community.

Distinctive receptor binding properties of the surface glycoprotein of a natural feline leukemia virus isolate with unusual disease spectrum.

BACKGROUND: Feline leukemia virus (FeLV)-945, a member of the FeLV-A subgroup, was previously isolated from a cohort of naturally infected cats. An unusual multicentric lymphoma of non-T-cell origin was observed in natural and experimental infection with FeLV-945. Previous studies implicated the FeLV-945 surface glycoprotein (SU) as a determinant of disease outcome by an as yet unknown mechanism. The present studies demonstrate that FeLV-945 SU confers distinctive properties of binding to the cell surface receptor. RESULTS: Virions bearing the FeLV-945 Env protein were observed to bind the cell surface receptor with significantly increased efficiency, as was soluble FeLV-945 SU protein, as compared to the corresponding virions or soluble protein from a prototype FeLV-A isolate. SU proteins cloned from other cohort isolates exhibited increased binding efficiency comparable to or greater than FeLV-945 SU. Mutational analysis implicated a domain containing variable region B (VRB) to be the major determinant of increased receptor binding, and identified a single residue, valine 186, to be responsible for the effect. CONCLUSIONS: The FeLV-945 SU protein binds its cell surface receptor, feTHTR1, with significantly greater efficiency than does that of prototype FeLV-A (FeLV-A/61E) when present on the surface of virus particles or in soluble form, demonstrating a 2-fold difference in the relative dissociation constant. The results implicate a single residue, valine 186, as the major determinant of increased binding affinity. Computational modeling suggests a molecular mechanism by which residue 186 interacts with the receptor-binding domain through residue glutamine 110 to effect increased binding affinity. Through its increased receptor binding affinity, FeLV-945 SU might function in pathogenesis by increasing the rate of virus entry and spread in vivo, or by facilitating entry into a novel target cell with a low receptor density.

MALT1 Inhibition Is Efficacious in Both Naïve and Ibrutinib-Resistant Chronic Lymphocytic Leukemia.

The clinical efficacy displayed by ibrutinib in chronic lymphocytic leukemia (CLL) has been challenged by the frequent emergence of resistant clones. The ibrutinib target, Bruton's tyrosine kinase (BTK), is essential for B-cell receptor signaling, and most resistant cases carry mutations in BTK or PLCG2, a downstream effector target of BTK. Recent findings show that MI-2, a small molecule inhibitor of the para-caspase MALT1, is effective in preclinical models of another type of BCR pathway-dependent lymphoma. We therefore studied the activity of MI-2 against CLL and ibrutinib-resistant CLL. Treatment of CLL cells in vitro with MI-2 inhibited MALT1 proteolytic activity reduced BCR and NF-κB signaling, inhibited nuclear translocation of RelB and p50, and decreased Bcl-xL levels. MI-2 selectively induced dose and time-dependent apoptosis in CLL cells, sparing normal B lymphocytes. Furthermore, MI-2 abrogated survival signals provided by stromal cells and BCR cross-linking and was effective against CLL cells harboring features associated with poor outcomes, including 17p deletion and unmutated IGHV Notably, MI-2 was effective against CLL cells collected from patients harboring mutations conferring resistance to ibrutinib. Overall, our findings provide a preclinical rationale for the clinical development of MALT1 inhibitors in CLL, in particular for ibrutinib-resistant forms of this disease. Cancer Res; 77(24); 7038-48. ©2017 AACR.

Disruption of pre-B-cell receptor signaling jams the WNT/ß-catenin pathway and induces cell death in B-cell acute lymphoblastic leukemia cell lines.

Targeting components of the B-cell receptor (BCR) pathway have dramatically improved clinical outcomes in a variety of B-cell malignancies. Despite the well-documented pathogenic role of BCR precursor (pre-BCR) pathway in B-cell acute lymphoblastic leukemia (B-ALL), there is limited available data of therapies that aim to disrupt this pathway. To investigate the role of protein kinase Cß (PKCß), a crucial mediator of BCR and pre-BCR signaling, in B-ALL survival, we studied the activity of the PKCß selective inhibitor enzastaurin (ENZ) in seven B-ALL cell lines. Treatment with ENZ resulted in a dose- and time-dependent growth inhibition in all cell lines with a relatively higher efficacy in pro-B ALL with translocation t(4;11)(q21;q23). The mechanism of growth inhibition was by apoptotic induction and cell cycle arrest. A rapid reduction in phosphorylation of AKT and its downstream target glycogen synthase kinase 3ß (GSK3ß) were observed at 30min after treatment and remaining for 48h. The reduction in GSK3ß phosphorylation was associated with a paradoxical accumulation of ß-catenin, which was due to a transient loss of ß-catenin phosphorylation at ser33-37. In addition, accumulation of ß-catenin was associated with downregulation of c-Myc, upregulatiuon of c-Jun, and a subsequent protective effect on the tumor suppressor p73. Data in this paper were presented in part at 2012 American Society of Hematology Annual Meeting, abstract 1350.

Insulin-like growth factor I reduces lipid oxidation and foam cell formation via downregulation of 12/15-lipoxygenase.

OBJECTIVE: We have shown that insulin-like growth factor I (IGF-1) infusion in Apoe(-/-) mice decreased atherosclerotic plaque size and plaque macrophage and lipid content suggesting that IGF-1 suppressed formation of macrophage-derived foam cells. Since 12/15-lipoxygenase (12/15-LOX) plays an important role in OxLDL and foam cell formation, we hypothesized that IGF-1 downregulates 12/15-LOX, thereby suppressing lipid oxidation and foam cell formation. APPROACH AND RESULTS: We found that IGF-1 decreased 12/15-LOX plaque immunopositivity and serum OxLDL levels in Apoe(-/-) mice. IGF-1 reduced 12/15-LOX protein and mRNA levels in cultured THP-1 macrophages and IGF-1 also decreased expression of STAT6 transcription factor. IGF-1 reduction in macrophage 12/15-LOX was mediated in part via a PI3 kinase- and STAT6-dependent transcriptional mechanism. IGF-1 suppressed THP-1 macrophage ability to oxidize lipids and form foam cells. IGF-1 downregulated 12/15-LOX in human blood-derived primary macrophages and IGF-1 decreased LDL oxidation induced by these cells. IGF-1 reduced LDL oxidation and formation of foam cells by wild type murine peritoneal macrophages, however these effects were completely blocked in 12/15-LOX-null macrophages suggesting that the ability of IGF-1 to reduce LDL oxidation and foam cells formation is dependent on its ability to downregulate 12/15-LOX. CONCLUSIONS: Overall our data demonstrate that IGF-1 reduces lipid oxidation and foam cell formation via downregulation of 12/15-LOX and this mechanism may play a major role in the anti-atherosclerotic effects of IGF-1.

Substitution of feline leukemia virus long terminal repeat sequences into murine leukemia virus alters the pattern of insertional activation and identifies new common insertion sites.

The recombinant retrovirus, MoFe2-MuLV (MoFe2), was constructed by replacing the U3 region of Moloney murine leukemia virus (M-MuLV) with homologous sequences from the FeLV-945 LTR. NIH/Swiss mice neonatally inoculated with MoFe2 developed T-cell lymphomas of immature thymocyte surface phenotype. MoFe2 integrated infrequently (0 to 9%) near common insertion sites (CISs) previously identified for either parent virus. Using three different strategies, CISs in MoFe2-induced tumors were identified at six loci, none of which had been previously reported as CISs in tumors induced by either parent virus in wild-type animals. Two of the newly identified CISs had not previously been implicated in lymphoma in any retrovirus model. One of these, designated 3-19, encodes the p101 regulatory subunit of phosphoinositide-3-kinase-gamma. The other, designated Rw1, is predicted to encode a protein that functions in the immune response to virus infection. Thus, substitution of FeLV-945 U3 sequences into the M-MuLV long terminal repeat (LTR) did not alter the target tissue for M-MuLV transformation but significantly altered the pattern of CIS utilization in the induction of T-cell lymphoma. These observations support a growing body of evidence that the distinctive sequence and/or structure of the retroviral LTR determines its pattern of insertional activation. The findings also demonstrate the oligoclonal nature of retrovirus-induced lymphomas by demonstrating proviral insertions at CISs in subdominant populations in the tumor mass. Finally, the findings demonstrate the utility of novel recombinant retroviruses such as MoFe2 to contribute new genes potentially relevant to the induction of lymphoid malignancy.

Unique long terminal repeat and surface glycoprotein gene sequences of feline leukemia virus as determinants of disease outcome.

The outcome of feline leukemia virus (FeLV) infection in nature is variable, including malignant, proliferative, and degenerative disorders. The determinants of disease outcome are not well understood but are thought to include viral, host, and environmental factors. In particular, genetic variations in the FeLV long terminal repeat (LTR) and SU gene have been linked to disease outcome. FeLV-945 was previously identified as a natural isolate predominant in non-T-cell neoplastic and nonneoplastic diseases in a geographic cohort. The FeLV-945 LTR was shown to contain unique repeat elements, including a 21-bp triplication downstream of the enhancer. The FeLV-945 SU gene was shown to encode mutational changes in functional domains of the protein. The present study details the outcomes of infection with recombinant FeLVs in which the LTR and envelope (env) gene of FeLV-945, or the LTR only, was substituted for homologous sequences in a horizontally transmissible prototype isolate, FeLV-A/61E. The results showed that the FeLV-945 LTR determined the kinetics of disease. Substitution of the FeLV-945 LTR into FeLV-A/61E resulted in a significantly more rapid disease onset but did not alter the tumorigenic spectrum. In contrast, substitution of both the FeLV-945 LTR and env gene changed the disease outcome entirely. Further, the impact of FeLV-945 env on the disease outcome was dependent on the route of inoculation. Since the TM genes of FeLV-945 and FeLV-A/61E are nearly identical but the SU genes differ significantly, FeLV-945 SU is implicated in the outcome. These findings identify the FeLV-945 LTR and SU gene as determinants of disease.

Feline leukaemia virus LTR variation and disease association in a geographical and temporal cluster.

Feline leukaemia virus (FeLV)-945 was previously identified in natural multicentric lymphomas and contains a 21 bp tandem triplication in the LTR. In the present study, FeLV LTR variation was examined in the cohort from which FeLV-945 was identified. The objectives of the study were to evaluate FeLV LTR variation within the cohort, to determine whether the FeLV-945 LTR was associated uniquely with multicentric lymphoma and to evaluate functional attributes that may have contributed selective advantage to the predominant LTR variants observed. T-cell tumours uniformly contained LTRs with duplicated enhancer sequences, although enhancer duplications conferred little transcriptional advantage. Non-T-cell malignant, proliferative and degenerative diseases contained LTRs with two, three or four tandemly repeated copies of the 21 bp sequence originally identified in FeLV-945. While the length and termini of enhancer duplications were variable, the 21 bp repeat unit was invariant. Triplication of the 21 bp repeat conferred the optimal replicative advantage in feline cells.