bluuebunny/crossref_metadata_2025 huggingface dataset:
IPI-504, a Novel, Orally Active HSP90 Inhibitor, Prolongs Survival of Mice with BCR-ABL T315I CML and B-ALL.
Authors: Peng Cong , Brain Julia , Hu Yiguo , Kong Linghong , Grayzel David , Pak Roger , Read Margaret , Li Shaoguang
Published: October 2019

Development of mutations within the kinase domain is a major drug-resistance mechanism for tyrosine kinase inhibitors (TKIs) in cancer therapy. In CML (chronic myeloid leukemia), a disease driven by the constitutively active BCR-ABL oncoprotein, no available TKIs have been effective in treating patients with the BCR-ABL T315I mutation. Heat shock protein 90 (Hsp90) is a highly conserved, constitutively expressed molecular chaperone that facilitates folding of client proteins like BCR-ABL, and affects the stability of these proteins. Several labs have shown that Hsp90 inhibition in vitro results in the degradation of BCR-ABL T315I and induces potent killing of these cell lines. However, these results have not been demonstrated in animal models for BCR-ABL-induced CML and B-ALL (B-cell acute lymphoblastic leukemia, a disease that does not respond well to TKIs including imatinib and dasatinib). Thus, IPI-504, an orally administered Hsp90 inhibitor, was evaluated in murine models of CML and B-ALL. Treatment of mice with wild type (WT)- or T315I BCR-ABL-induced CML with IPI-504 resulted in BCR-ABL protein degradation and a decrease in circulating BCR-ABL positive cells. In response to treatment with vehicle the median survival time of WT and T315I CML mice is approximately 20 days. While the T315I CML mice were resistant to imatinib with a median survival of 21 days, IPI-504 (50 and 100 mg/kg, PO TIW) demonstrated dose-dependent prolonged survival of these mice by 30 and 70 days, respectively (p<0.001 for both doses). Both imatinib and IPI-504 similarly prolonged survival of mice with BCR-ABL-WT-induced CML. In the T315I CML mice prolonged survival of the IPI-504 treated cohort was associated with decreased peripheral blood BCR-ABL positive leukemia cells during treatment, less splenomegaly and improved pulmonary histopathlogy at necropsy. In CML mice receiving mixed BCR-ABL-WT- or T315I-transduced donor bone marrow cells, Hsp90 inhibition more potently suppressed T315I-expressing leukemia clones relative to the WT clones, consistent with in vitro studies where T315I BCR-ABL was more sensitive to IPI-504 induced degradation in cell lines than WT BCR-ABL. Combination treatment with IPI-504 and imatinib was more effective than either treatment alone in prolonging survival of mice bearing both WT and T315I leukemic cells. IPI-504 also significantly prolonged survival of B-ALL mice bearing the T315I mutation (p<0.001).

These results provide a rationale for use of an Hsp90 inhibitor as a novel approach to overcoming resistance to TKIs as well as the potential for first line combination treatment in CML patients. The potential for IPI-504 to eliminate mutant kinases via Hsp90 inhibition provides a new therapeutic strategy for treating BCR-ABL-induced CML, ALL as well as other cancers resistant to treatment with TKIs.
The Cryptic Chromosomal Deletion, del(11)(p12p13), as a New Activation Mechanism of LMO2 in Pediatric T-Cell Acute Lymphoblastic Leukemia.
Authors: Van Vlierberghe Pieter , van Grotel Martine , Beverloo H. Berna , Lee Charles , Helgason Tryggvi , Buijs-Gladdines Jessica , Passier Monique , van Wering Elisabeth R. , Veerman Anjo J.P. , Kamps Willem A. , Meijerink Jules P.P. , Pieters Rob
Published: October 2019

To identify new cytogenetic abnormalities associated with leukemogenesis or disease outcome, T-ALL patient samples were analyzed by means of the array-comparative genome hybridization technique (array-CGH). Here, we report the identification of a new recurrent and cryptic deletion on chromosome 11, i.e. the del(11)(p12p13), in about 4% (6/138) of pediatric T-ALL patients. Detailed molecular-cytogenetic analysis revealed that this deletion activates the LMO2 oncogene in 4 out of 6 del(11)(p12p13) positive T-ALL patients, alike patients with an LMO2 translocation (9/138). The LMO2 activation mechanism of this deletion is loss of a negative regulatory region upstream of LMO2, causing activation of the proximal LMO2 promoter. LMO2 rearrangements, including this del(11)(p12p13) and t(11;14)(p13;q11) or t(7;11)(q35;p13), were found in the absence of other recurrent cytogenetic abnormalities involving HOX11L2, HOX11, CALM-AF10, TAL1, MLL or MYC. LMO2 abnormalities, represent about 9% (13/138) of pediatric T-ALL cases, and are more frequent in pediatric T-ALL than appreciated up till now.
Imatinib Inhibits Both CD4+ T Regulatory Cells and CD8+ T Lymphocytes Specifically Directed Against the Leukemia-Associated Antigen RHAMM/CD168.
Authors: Chen Jinfei , Schmitt Anita , Chen Baoan , Rojewski Markus , Greiner Jochen , Guillaume Philippe , Dohner Hartmut , Bunjes Donald , Schmitt Michael
Published: October 2019

Imatinib mesylate (IM, STI571, Gleevec®) is highly effective in the treatment of patients with chronic myelogenous leukemia (CML). In patients with residual disease or relapse after allogeneic stem cell transplantation (allo-SCT), the optimal strategy is a matter of debate: donor lymphocyte infusions (DLIs) alone, IM alone or a combination of both have been administered to CML patients in this clinical situation. As an impairment of anti-viral CD8+ T lymphocyte function by IM has been described, we questioned whether IM also affects specific anti-leukemic CD8+ T lymphocytes. Moreover, we asked to which extent CD4+CD25hi T regulatory cells might be affected by IM thus changing the balance between cytotoxic and tolerogenic T cells. After mixed lymphocyte peptide culture (MLPC), we assessed CD8+ T cells from healthy donors and patients with CML after allo-SCT by tetramer staining/multi-color flow cytometry and enzyme linked immunosorbent spot (ELISPOT) assays, as well as CD4+CD25hicells after 3 days of culture with anti-CD3 and anti-CD28. The release of interferon gamma and granzyme B by CD8+ T lymphocytes specific for R3 (ILSLELMKL), a T cell epitope peptide derived from the leukemia-associated antigen designated RHAMM/CD168 (receptor for hyaluronic acid mediated motility) was inhibited by IM in a clearly dose-dependent fashion in a range from 1 to 25 μM. These T cells were further characterized as CD8+ HLA-A2/R3_tetramer+ WT1_tetra-CD45RA+CD27-CD28-CCR7- effector T cells with the ability to lyse R3 peptide labeled T2 cells and CD34+ CML progenitor cells. The inhibition of CD8+ T lymphocytes was a function of the time of exposure to IM and reversible after removal of IM from the MLPC. On the other hand, the proliferation and function of CD4+CD25hiFoxP3+GITR+TGFß1+ CD69+CD152+ T regulatory cells were also significantly inhibited by IM in a dose-related fashion. These findings suggest that the clinical impact IM must not necessarily result in a reduced efficacy of the graft-versus-leukemia effect observed after allo-SCT and/or DLIs but might depend rather on the ratio of CD8+ cytotoxic T cells and CD4+CD25hi T regulatory cells.
Hydroxamate Pan-HDAC Inhibitor LBH589 Depletes EZH2 and DNMT1, Partly through hsp90 Inhibition and Its Chaperone Association with DNMT1.
Authors: Fiskus Warren , Herger Bryan , Rao Rekha , Atadja Peter , Bhalla Kapil
Published: October 2019

The PRC (polycomb repressive complex) 2 is a multi-protein complex, which includes EZH2, SUZ12 and EED proteins. The PRC2 complex possesses histone methyltransferase (HMTase) activity mediated by the SET domain of EZH2, which methylates histone H3 on lysine (K)-27. We have previously reported that the treatment with the hydroxamate, pan-HDAC inhibitor LBH589 acetylates and inhibits the ATP binding and chaperone function of hsp90, as well as depletes the levels of EZH2, Suz12 and EED in AML cells (Blood106 (11) 2482, 2005). Recently, within the PRC2, EZH2 was shown to interact with and modulate the DNA methyltransferases DNMT1, DNMT3a and DNMT3b, which affects their binding to the EZH2-targeted gene promoters. Thus EZH2 may serve as a recruitment platform for DNA methyltransferases, thereby mechanistically linking the two epigenetic silencing systems, maintaining the chromatin in the silenced state and inhibiting gene expression. In the present studies we determined the effects of hydroxamate pan-HDAC inhibitor LBH589 on the interaction between the PRC2 proteins EZH2 and EED and DNMT1 in CML blast crisis (CML-BC) K562 and LAMA-84 cells. Treatment with ≥50 nM LBH589 for 24 hours disrupted the interaction between DNMT1 and Ezh2, as well as depleted the levels of DNMT1 in a dose and time dependent manner. LBH589 (≥50 nM) also disrupted the binding of DNMT1 to hsp90. This was associated with increased polyubiquitylation and accumulation of DNMT1 in the detergent insoluble fraction of the leukemia cells. Notably, similar findings were obtained following treatment with the hsp90 inhibitor 17-DMAG (250 nM). Taken together, these findings suggest that DNMT1 has chaperone association with hsp90, which is disrupted by LBH589. Additionally, RT-PCR analysis demonstrated that treatment with 100 nM LBH589 for 8 hours depleted the mRNA expression of DNMT1 in K562 cells, suggesting that both transcriptional and post-transcriptional mechanisms are involved in LBH589 mediated down regulation of DNMT1. Depletion of DNMT1 due to LBH589 treatment was associated with induction of JunB mRNA expression. This may be because JunB is repressed by DNA hypermethylation in CML-BC cells. A similar upregulation of JunB mRNA and protein in K562 cells also followed shRNA mediated depletion of DNMT1 (down to 40%). These findings indicate that treatment with pan-HDAC inhibitor LBH589 not only induces histone acetylation and depletes the levels of histone methyl transferase EZH2, but also down regulates DNMT1 through transcriptional mechanism and proteasome degradation. The latter effect may be due to LBH589 mediated acetylation and inhibition of chaperone function of hsp90, an effect also seen with treatment with 17-DMAG.
Constitutive Activation of Toll like Receptor Signaling in Adult T-Cell Leukemia Cells.
Authors: Mizobe Takamitsu , Tsukada Junichi , Higashi Takehiro , Mouri Fumihiko , Matsuura Ai , Tanaka Yoshiya
Published: October 2019

Human T-cell leukemia virus type I (HTLV-I) is etiologically associated with the development of an aggressive and fatal malignancy of CD4+ T lymphocytes called adult T-cell leukemia (ATL). Constitutive activation of nuclear factor-κB (NF-κB) is a common feature of ATL. Although the mechanism by which NF-κB is spontaneously activated in ATL cells still remains unclear, inhibition of NF-κB activity induces apoptosis, suggesting a central role of NF-κB in their proliferation. Toll like receptors (TLRs) are involved in innate cell activation by conserved structures expressed by microorganisms. Engagement of IL-1R or TLR with their cognate ligands causes an adaptor protein MyD88 to be recruited to the receptor complex, which in turn promotes its association with the IL-1R-associated kinase (IRAK) via an interaction between the respective death domains of each molecule. Several recent reports have indicated unique expression profiles of TLRs on different subsets of human T cells, and that some TLR ligands modulate the function of human T cells. We examined expression of the TLR mRNAs in primary ATL cells and ATL cell lines, MT2, MT4 and HUT102 by RT-PCR. Expression of TLR mRNAs, except of TLR7 and TLR8, was detected in all cell samples examined. We further demonstrated constitutive association of MyD88, an adaptor protein for the TLR signaling, with the IL-1R-associated kinase 1 (IRAK1) in ATL cell lines, MT2, MT4 and HUT102. In MT2 cells, constitutive activation of NF-κB and NF-IL6, but not Stat3 was significantly inhibited by expression of a dominant negative form of MyD88 protein (MyD88dn). Spontaneous transcriptional activation of IL-1α, IFN-γ and TNF-α gene promoters in MT2 cells was also suppressed by MyD88dn expression. MyD88dn inhibited cell proliferation and induced apoptosis of MT2 cells. In addition, overexpression of wild-type MyD88 and HTLV-I Tax induces synergistically transcriptional activity of NF-κB in 293T cells, showing interaction of Tax with MyD88. Thus, our results show a critical role of MyD88 in dysregulated gene activation and cell proliferation in HTLV-I-transformed T-cells, and further suggest the involvement of MyD88 in Tax-mediated intracellular signal transduction in HTLV-I-infected cells. Considering the fact that blocking NF-κB is a potential strategy to treat ATL, our argument raises a possibility that we may be able to find new treatment targets against ATL.
Imidazoacridinones Are Bifunctional Targeting Agents Active in Leukemia Cells.
Authors: Chau MyDoanh , Otake Yoko , Stuart Robert K. , Fernades Daniel J. , Ajami Alfred M.
Published: October 2019

C-1311 (Symadex™) is the lead compound in clinical development from a new series of agents, the imidazoacridinones. It was previously reported that C-1311 was solely a topoisomerase II (TOP2) cleavable complex inhibitor and DNA damaging agent. However, contemporary analyses of C-1311 point to alternative structural parallelisms with the molecular scaffolds now seen as common motifs in receptor tyrosine kinase (RTK) inhibitors. To investigate this mechanism of action, a kinase inhibitor screening program was initiated, first in silico by molecular similarity matching, and then in vitro against recombinant RTKs. C-1311 was identified to cluster with RTK inhibitors, especially when sunitinib is used as a molecular “seed” for pattern recognition. The statistical similarity coefficients fall in the 0.7–0.8 positive probability range, compared to 0.2–0.4 for the classical TOP2 inhibitors. C-1311 was found to selectively inhibit wild-type (WT) FLT3 and its D835Y mutant at nanomolar concentrations of 8–15 nM IC50. FGFR2 was also targeted at approximately 100-fold greater IC50 value of 1.2 μM. Inhibition of PDGFR, FGFR1, and KIT was observed at about 3–4 μM IC50; other prominent kinases had values of greater than 10 μM. As a part of the dual mechanism reexamination, TOP2 decatenation of kDNA, DNA relaxation, and competition assays were performed to assess C-1311 interaction with TOP2. Results confirmed that C-1311 is not a cleavable complex inhibitor when compared to etoposide, suggesting that it may not be an inducer of the MLL cleavage. Additionally, the IC50 of C-1311 from the decatenation assay is 44 μM compared to 3, 12, and 149 μM for mitoxantrone, daunorubicin, and merbarone, respectively. Cellular screens were performed to assess the potency of C-1311 in myeloid and lymphoid leukemia cell lines. C-1311 was equally effective in both WT FLT3 expressing (RS4;11) and mutant FLT3 expressing (MV-4–11) cell lines. The dual function of C-1311 may contribute to the potency in these cell lines. These findings warrant further investigation of C-1311 in yet unexplored indications, including other hematological malignancies.
Abnormalities in Glucose Uptake and Metabolism in Imatinib-Resistant Bcr-Abl Positive Cells.
Authors: Serkova Natalie J. , Kominsky Douglas J. , Brown Jaimi L. , Miljus Jelena , Boros Laszlo G. , Eckhardt Gail S. , Melo Junia V.
Published: October 2019

The development of imatinib resistance has become a significant therapeutic problem, in which the etiology appears to be multifactoral (Bcr-Abl mutation and/or overexpression, multidrug-resistance, protein overexpression, etc.) and poorly understood. As of today, there are no precise clinical criteria to predict the development of imatinib resistance in CML, other than relapse of the disease. However, there is evidence that the control of glucose-substrate flux is an important mechanism of the antiproliferative action of imatinib. Thus, imatinib-resistant gastrointestinal stromal (GIST) KIT-activated tumors reveal highly elevated glucose uptake in PET scans. Currently, there is no information about the changes in glucose cell metabolism during development of imatinib resistance in CML patients. We used nuclear magnetic resonance (NMR) spectroscopy and gas chromatography mass spectrometry (GC-MS) to assess 13C glucose uptake and metabolism (glycolysis, the TCA cycle, nucleic acid ribose synthesis) during imatinib treatment in cultured human CML cells with different sensitivity to imatinib. Our results demonstrate that imatinib-sensitive K562 and LAMA84 Bcr-Abl positive cells have decreased glucose uptake, decreased lactate production and improved oxidative TCA cycle upon imatinib treatment. The LAMA84-r (resistant to imatinib due to Bcr-Abl overexpression) and the K562-r (unknown mechanism of resistance) cells maintained high glycolytic metabolic phenotype with elevated glucose uptake and lactate production in the presence of imatinib (Figure 1). In addition, oxidative synthesis of RNA ribose from 13C-glucose via glucose-6-phosphate dehydrogenase was decreased and RNA synthesis via the non-oxidative transketolase pathway was increased in imatinib resistant cells. CML cells which exhibited an [oxidative/non-oxidative] flux ratio for nucleic acid ribose synthesis > 1, were sensitive to imatinib. The resistant K562-r and LAMA84-r exhibited the [oxidative/non-oxidative] flux ratio < 0.7. We correlated 13C glucose uptake and metabolism with intracellular expression and localization of GLUT-1 transporter protein in these cells. There was no difference in total protein load of GLUT-1 upon imatinib treatment between imatinib-sensitive and resistant cell lines. However, the changes in glucose uptake and metabolism were accompanied by intracellular trans-location of GLUT-1 from the plasma membrane to the cytoplasm in imatinib-sensitive treated cells, while GLUT-1 remained at the membrane surface in LAMA84-r and K562-r cells (Figure 1). In summary, elevated glucose uptake and non-oxidative glycolytic metabolic phenotype can be used as sensitive markers for imatinib resistance in Bcr-Abl positive cells with different origins of resistance.

Figure 1: Figure 1:.
VEGFR-1 (FLT-1), b1 Integrin and hERG K+ Channel Form a Macromolecular Signaling Complex in Acute Myeloid Leukemia Cells.
Authors: Arcangeli Annarosa , Pillozzi Serena , Brizzi Maria F. , Bernabei Pietro A. , Bartolozzi Benedetta , Caporale Roberto , Basile Venere , Boddi Vieri , Torre Eugenio , Pegoraro Luigi , Becchetti Andrea
Published: October 2019

Acute myeloid leukemia (AML) is a heterogeneous group of clonal bone marrow (BM) malignancies, presenting a wide spectrum of morphologic, immunophenotypic, cytogenetic, and molecular features. There is general agreement that persistent detection of leukemia cells outside the BM microenvironment, especially when accompanied by infiltration into extramedullary sites, is a clear negative prognostic factor. Infiltration may also reduce leukemia responsiveness to induction chemotherapy. Great attention is thus being played to the molecular mechanisms that regulate acute leukemia cells exit from the BM. A crucial role is thought to be played by the angiogenic factors and angiogenesis-related signals, that operate inside the BM microenvironment.The persistence of circulating leukemia blasts and their engraftment into extramedullary sites are negative prognostic factors, in patients with acute leukemia. Leukemia cell exit from the bone marrow is regulated by angiogenic factors and in particular by the Vascular Endothelial Growth Factor and its receptor 1, FLT-1. We have studied the cross talk between

FLT-1, the human eag-related gene 1 (hERG1) K+ channels, integrin receptors, in acute myeloid leukemia (AML) cells.

FLT-1, hERG1 and the b1 integrin were found to form a macromolecular signaling complex. The latter mostly recruited the hERG1B isoform of hERG1 channels, and its assembly was necessary for subsequent FLT-1 tyrosine phosphorylation and sustained AML cell migration. What is more, both effects were inhibited when hERG1 channel were specifically blocked. The formation of an FLT-1/hERG1/b1 complex was also observed in primary AML blasts, obtained from a population of human patients. The coexpression of FLT-1 and hERG1 conferred a pro-migratory phenotype to these AML blasts. Such a phenotype was also observed in vivo. Herg1 positive blasts were more efficient in invading the peripheral circulation and the extramedullary sites after engraftment into immunodeficient mice. Moreover, herg1 expression in leukemia patients correlated with a shorter overall survival. Overall, we conclude that hERG1 channels mediate the FLT-1 dependent AML cell migration and that their expression is a negative prognostic factor, in AML.
C-Terminal Deletion Mutation of CD20 in B-Cell Malignancy Is Related to a Risk of Relapse and Resistance to Chimeric Anti-CD20 Antibody Therapy.
Authors: Terui Yasuhito , Mishima Yuji , Sugimura Natsuhiko , Kojima Kiyotsugu , Kuniyoshi Ryoko , Rokudai Akiko , Mishima Yuko , Yokoyama Masahiro , Takeuchi Kengo , Hatake Kiyohiko
Published: October 2019

Introduction: Rituximab was the first chimeric antibody that is most frequently used for CD20-posistive B-cell lymphomas and gives better response and prognosis. However, resistance to rituximab is one of the important issues to be clarified in the increasing number of monoclonal antibody therapy, and we experienced the case whose lymphoma cells never expressed CD20 at the relapsed phase during rithuximab therapy. We investigated whether CD20 mutation is related to expression level of CD20, relapse or resistance to rituximab therapy, and prognosis.

Methods: We analyzed 50 patients with fresh or relapsed/ resistant B- cell lymphomas and DNA sequencing analysis of CD20 products from genomic PCR and RT-PCR was performed. CD20 mutants were subcloned by TA cloning, and tested CD20 expression after transfection to K562 cells.

RESULTS: Four types of CD20 mutations were found in 11 of 50 NHL patients (22.0%), which include C-terminal deletion(8.0%) extracellular domain (2.0%) transmembrane domain (2.0%) and early termination (10.0%). The group of C-terminal deletion mutations significantly showed lower CD20 expression (3.26, 95%CI = 0.09 to 6.89) than non-mutation (30.8, 95%CI = 22.4 to 39.2) (p < 0.05), whereas five patients with early termination mutation did not show significant difference of CD20 expression (19.5, 95%CI = 10.7 to 28.4) as compared with non-mutation (p > 0.05). Although there was no significant difference between the groups with non-mutation and C-terminal deletion mutation in CR rate (49% versus 25%, Fisher’s exact test; p = 0.6137), time to progression after rituximab therapy in C-terminal deletion mutation (7 months, 95%CI = 0 to 18 months) was significantly shorter than that in non-mutation (31 months, 95%CI = 18 to 44 months) by log-rank test (p = 0.0481).

Conclusion: The important mutations of CD20 gene related to shorter duration to progression disease after rituximab therapy were discovered. Especially, point mutations bearing during rituximab therapy should be examined at progression disease after partial remission.
Rituximab-Mediated Inhibition of the p38 MAPK and NF-κB Survival Pathways In Vitro and Validation in Nude Mice Bearing the Raji B-NHL Tumor.
Authors: Vega Mario I. , Huerta-Yepez Sara , Martinez-Paniagua Melisa A. , Bonilla Cesar G. , Bonavida Benjamin
Published: October 2019

Treatment of B-NHL cell lines with rituximab inhibited the p38 MAPK and NF-κB pathways, resulting in chemosensitization to drug-induced apoptosis (Vega et al., 2004 Oncogene, 23:3530–40; Jazirehi et al., 2005 Cancer Res., 65:264–76). Chemosensitization was the direct result of inhibition by these pathways of the anti-apoptotic gene products Bcl-2/Bcl-xL. Cell signaling by rituximab in B-NHL patients has not been investigated and thus, we have examined an in vivo model bearing a tumor xenograft for validation. Balb/c nude mice were transplanted s.c. with the B-NHL cell line Raji; a group of mice was left untreated and another group was treated with rituximab (1000 μg) at days 5 and 10 following implantation of 8x106 tumor cells. The animals were monitored for tumor cell growth and sacrificed at day 30 and tumors were obtained for further analysis. Treatment with rituximab resulted in significant inhibition of tumor cell growth compared to control. Tumor tissues were examined by immunohistochemistry for phospho and non-phospho p38 MAPK and NF-κB (p50), Bcl-2 and Bcl-xL expression. Analysis of tumor-derived tissues from control mice revealed overexpression of phospho-p38 MAPK and NF-κB (p50) and strong nuclear localization of phospho-p50. In addition, there was overexpression of Bcl-2 and Bcl-xL. In contrast, tumor tissues derived from rituximab-treated mice demonstrated significant inhibition of phospho-p38 MAPK, phospho-p50-NF-κB and reduced nuclear localization of phospho-p50. In addition, Bcl-2 and Bcl-xL expression was also significantly reduced. These findings established for the first time, in a pre-clinical model, rituximab-mediated inhibition of the cell survival pathways mediated by p38 MAPK and NF-κB. In addition, the study also corroborates the role of Bcl-2 and Bcl-xL expression in resistance and their inhibition by rituximab. In a separate abstract, tumor tissues derived from many untreated B-NHL patients were analyzed by immunohistochemistry for the activation of both phospho-p38 MAPK and NF-κB and compared to biopsies derived from control individuals. Overall, this study suggests that the p38 MAPK and NF-κB survival pathways are targets for rituximab-mediated effects and also suggests that such targets can be used for intervention in cases of rituximab resistance.
The PIG-A Gene Is Hypermutable in Lymphoid Neoplasms.
Authors: DiTata Kimberly , Araten David J.
Published: October 2019

In neoplasms, the presence of chromosomal abnormalities and point mutations is suggestive of genomic instability, but could also be a consequence of selection. While genomic instability may increase the chances that a malignant population acquires adaptive mutations, extremely high mutation rates may not be compatible with cell survival. To investigate the role of genomic instability in lymphoid neoplasms, we have applied a new method for the quantification of the human mutation rate, using the PIG-A gene as a sentinel. PIG-A is essential for the biosynthesis of glycosylphosphatidylinositol (GPI) and is mutated in blood cells of patients with Paroxysmal Nocturnal Hemoglobinuria. A broad range of mutations can produce the GPI (−) phenotype, and because PIG-A is on the X-chromosome, the effect of a single mutation is detectable. Since a host of proteins require GPI for attachment to the cell surface, rare mutants are readily detected by flow cytometry. We have previously shown that PIG-A mutations arise spontaneously in normal donors, and we determined that the mutation rate in normal B cell lines ranges from 2 to 29 per 107 cell divisions. Here we analyzed cell lines derived from:

a transformed low grade lymphoma harboring a t(14;18) translocation; a mantle cell lymphoma harboring a t(11;14) translocation; a marginal zone lymphoma; and T cell ALL.

Cells were first stained with an antibody specific for CD59 (a representative GPI-linked protein) and pre-existing GPI (−) cells were eliminated from the population by flow cytometric sorting, by gating on the upper 50th percentile of the distribution curve. The collected GPI (+) cells were then returned to culture and the number of cell divisions (d) determined by cell counts. After 3–4 weeks, the frequency (f) of new mutants arising in culture was determined by flow cytometric analysis of a large number of cells (median 2.3 x 106). Cells were stained simultaneously with antibodies specific for at least 3 GPI-linked proteins (e.g. CD48, CD52, CD55, and CD59) as well as a transmembrane protein (e.g. HLA-DR or CD45) to identify live cells. FLAER and proaerolysin-- which bind to GPI-- were used to confirm the phenotype. The frequency of mutants was determined by the number of GPI(−) cells divided by the number of GPI(+) cells analyzed, and the mutation rate (μ) was calculated with the formula μ = f ÷ d. We demonstrated a high mutation rate in 3 out of the 4 cells lines: 1750 x 10−7 (transformed lymphoma), 335 x 10−7 (mantle cell lymphoma), 112 x 10−7 (T cell ALL). Of note, the mutation rate was normal (4 x 10−7) in the marginal zone lymphoma—consistent with this being an indolent neoplasm. These data support the hypothesis that an elevated mutation rate is part and parcel of aggressive neoplasms and demonstrate that a 2-log elevation in this parameter is compatible with cell survival. With this model, it may be possible to predict the development of mutations that confer chemotherapy drug resistance.
Evidence for Non-Random Immunoglobulin VH Gene and Light Chain Isotype Usage in Follicular Non-Hodgkin’s Lymphoma.
Authors: Yohn Christopher B. , Van Beveren Charles P. , Mu Xi Y. , Shier Peter , Silverman Gregg J. , Gold Daniel P.
Published: October 2019

Background: Antibody diversity is generated by recombination of individual immunoglobulin (Ig) gene segments and subsequent somatic diversification driven by antigen recognition. In the repertoire of expressed B cell receptors (BCR) among normal peripheral B cells, variable heavy (VH) gene segments are not equally represented. The ratio of kappa to lambda light chain usage is also skewed; the normal κ/λ is 1.5. Investigation of the BCR repertoire may provide clues to the genesis of B cell malignancies, as suggested in chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL). BCR V gene identification during the production of recombinant antibodies used in our ongoing PhII and PhIII FavId® (idiotype/KLH) immunotherapy studies has enabled us to analyze V gene usage from 475 B cell follicular lymphoma (FL) tissue samples. This study reports the results of VH gene and κ/λ gene expression in this FL sample collection.

Methods: Ig heavy chain (HC) and light chain (LC) isotypes from B cell FL samples were identified by flow cytometry. VH and VL regions were sequenced from gene specific cDNA libraries prepared from these samples. VH gene usage and κ/λ ratios were compared to frequencies determined for normal peripheral B cells isolated from six healthy volunteers as well as published reports for normal peripheral B cells and other B cell malignancies.

Results: Compared to VH gene family usage determined for normal B cells, VH3 usage is higher (68% vs. 42%), VH1 usage is lower (7.8% vs. 22%) and VH4 usage is equivalent (22% vs. 26%) in our cohort of FL patients while VH2, 5, 6 and 7 are infrequently used in both populations. Usage of the VH3 genes within FL derived sequences also depends upon isotype, in that this gene family is preferentially associated with the IgM HC isotype relative to IgG (76% and 57% respectively). Additionally, the combined usage of the specific genes VH3-23 and VH3-48 in our patient collection accounts for over 29% of all VH genes - compared to 9% among normal B cells. These VH gene usages also differ from reports of VH gene expression among CLL and MCL patients. With respect to LC usage, VH3 isolates are associated with a normal κ/λ ratio of 1.6 while VH4 gene isolates are preferentially associated with λ light chains with a κ/λ ratio of 0.9. Finally, FL B cells expressing the IgM HC isotype preferentially co-express κ light chains (κ/λ ratio of 2.4) while IgG expressing cells preferentially utilize λ chains (κ/λ ratio of 0.6).

Conclusions: Non-random V gene and LC expression among patients with FL is noted. These distortions in Ig gene expression suggest that lymphomagenesis in FL may be associated with B cell stimulation by common antigens. A program to investigate the epitopes recognized by FL derived BCRs via binding of recombinant FL derived antibodies to protein arrays containing common auto-antigens is currently underway.
Transmission of a Follicular Lymphoma by Allogeneic Bone Marrow Tranplantation — Evidence to Support the Existence of a Lymphoma Progenitor Cell.
Authors: Hart Jason , Turner A. Robert , Larratt Loree M. , Russell James A. , Franko Bevin , Frantz Christine , Paonessa Teresa , Mansoor Adnan , Lai Raymond
Published: October 2019

Background: Lymphomagenesis is believed to depend on clonal proliferation of neoplastic progenitor cells. For follicular lymphoma, the neoplastic progenitors are likely derived from the early B-cells, but their exact nature remains elusive. We report a case of transmission of a follicular lymphoma by allogeneic bone marrow transplantation. Follicular lymphoma developed in the donor, and subsequently in the recipient, years after the BMT. Testing supports a common progenitor cell for both the malignancies.

Methods: Sequencing of the amplifiable translocation (14;18) BCL2/IgH fusion gene product and microsatellite analysis using a commercially available human identity testing kit were used to establish the biological relatedness of the donor and recipient lymphomas. The monoclonal IgH gene rearrangements from these two lymphomas were further examined for their somatic hypermutational status.

Results: Microsatellite analysis confirmed the recipient’s lymphoma was donor cell-derived. Analysis of the monoclonal IgH gene rearrangements revealed post-germinal center cell phenotype in both tumors. The VH regions utilized were different in the donor and recipient lymphoma, demonstrating two distinct clonal populations. Despite this difference, the sequences of the translocation (14;18) fusion gene products (BCL2/IgH) from the two lymphomas were identical confirming a common progenitor cell for both malignancies.

Conclusion: Our findings strongly suggest the same neoplastic progenitor cells are responsible for the lymphomagenesis in both the donor and recipient. These findings support the concept of a lymphoma progenitor cell.
A Vascular Targeted Pan PI-3 Kinase Inhibitor, SF1126 with Activity Against Multiple Myeloma In Vivo.
Authors: De Pradip , Peng Qiong , Dey Nandini , McDermitt Breanne , Peng Xiaodong , Garlich Joseph , Lonial Sagar , Durden Donald L.
Published: October 2019

Background: Considerable evidence suggests an important role for the PI-3 kinase and AKT signaling pathways in survival and chemoresistance in multiple myeloma (MM) and other lymphoid malignancies. Our group and others have demonstrated that downregulation of p-AKT with combination therapy (bortezomib + lonafarnib; David et al, Blood, 2005) is a surrogate marker for myeloma apoptosis. It has been demonstrated that the compound, LY294002 has significant pan PI-3 kinase inhibitory properties but is not suitable for clinical use due to PK issues. SF1126 is a novel RGD targeted derivative of LY294002 that has been shown to have activity in a number of different tumor models. Herein, we evaluated the activity of SF1126 against the MM.1S and MM.1R MM cell lines in vitro and in vivo for sensitivity to PI-3 kinase inhibition. The results demonstrate that MM.1S and MM.1R tumor cell growth is sensitive to SF1126 with IC50 of 7.5 and 10.8 uM, respectively. The effects of SF1126 on MM.1R signaling in vitro was examined with profound inhibition of HIF1a induction under hypoxia, the suppression of phosphorylation states of MDM2, ERK and RS6 kinase. The IC50 for inhibition of p-AKT in MM.1S and MM.1R cells was determined to be 2.4 and 2.8 uM, respectively. SF1126 treatment (50 mg/kg/dose sc given every other day) inhibited MM.1R tumor growth in nude mouse xenografts 95% as compared to untreated controls on day 38 (p < .01). Microvessel density analysis of MM.1R tumor tissue demonstrated that SF1126 had significant antiangiogenic activity in vivo.

Conclusion: The results provide preclinical data to support SF1126 as a clinically viable antiangiogenic, pan PI-3 kinase inhibitor for Phase I clinical trials in the treatment of multiple myeloma. Further studies in primary myeloma cells and in combination with conventional agents will be presented.
A Phase II Study of Denileukin Diftitox (Ontak®) with CHOP Chemotherapy in Patients with Newly-Diagnosed Aggressive T-Cell Lymphomas, the CONCEPT Trial: Interim Analysis.
Authors: Foss Francine M. , Sjak-Shie Nelida , Goy Andre , Advani Ranjana , Jacobsen Eric , Acosta Mark
Published: October 2019

Denileukin diftitox, a genetically engineered fusion protein comprised of the enzymatically active domain of diphtheria toxin and the full length sequence of human interleukin-2 (IL-2) that targets malignancies expressing the IL-2 receptor, is approved for cutaneous T-cell lymphoma and has demonstrated a 48% objective response rate in relapsed/refractory PTCL. Because denileukin diftitox is not myelosuppressive and induces apoptosis by inhibition of protein synthesis, a mechanism of action non-cross-resistant with chemotherapy, we evaluated the efficacy and safety of the combination of denileukin diftitox plus CHOP in newly diagnosed aggressive T-cell lymphomas. In this multicenter Phase II study, patients (pts) with aggressive T-cell histologies according to REAL classification were eligible. Pts were required to have absolute neutrophil count ≥1000, platelets ≥50,000, adequate renal and hepatic function with serum albumin ≥3.0 g/dL, and LVEF ≥50%. Denileukin diftitox was given by IV infusion at 18 mcg/kg/day on Days 1 and 2 of each cycle, followed by CHOP chemotherapy on Day 3, and G-CSF support starting Day 4 of each cycle. Cycles were administered every 3 weeks for up to 6 cycles. Restaging evaluation of response is performed after every 2 cycles. The study objective for efficacy is to achieve an overall response rate (CR+PR) of 70%. Twenty-one patients (76% PTCLnos) have been enrolled to date, with a median age of 56 (range 40–80), 10 male, and 11 female. Six patients are not evaluable for response; one discontinued therapy during Cycle 1 due to a hypersensitivity reaction, and 5 are too early to evaluate (1 cycle of therapy given). For the 15 evaluable patients, the overall response rate is 87% with 9 CR or CRu (60%), 4 PR (27%) and 1 each for stable and progressive disease (see Table below). Four of 13 responders (31%) have progressed, with duration of response ranging from 1.5 to 5.9 months. Toxicities have generally been grade 1–2, were transient, and caused few treatment delays. The most common adverse events have been edema (27%), elevated transaminases (20%), and hypoalbuminemia (14%). Only two Grade 4 toxicities were noted (one each of neutropenia and deniluekin diftitox infusion-related allergic reaction). This is the first trial to report on the use of ONTAK in combination with chemotherapy. The novel combination of denileukin diftitox and CHOP appears to be clinically active, and is well tolerated with no unexpected, new, or overlapping toxicities. Pt accrual is ongoing.

PTCL subtype No. of Patients No. evaluable Responses ORR PTCLnos 16 12 4 CR 4 CRu, 3 PR, 1 PD 92% Angioimmunoblastic 2 1 1 PR 50% Enteric T-cell 2 2 1 CR, 1 SD 50% Subcutaneous panniculitic 1 0 n/a n/a
Cutaneous T Cell Lymphoma: Responses in Phase 1 Trial of Combination Therapy with Liposomal Doxorubicin, Bortezomib, and Gemcitabine.
Authors: Dhillon Navneet , Bakkannagari Srinivasa , Ng Chaan , Lim Joann , Duvic Madeline , Kurzrock Razelle
Published: October 2019

BACKGROUND: There is preclinical data suggesting pro-apoptotic synergy between the proteosome inhibitor bortezomib, doxorubucin and gemcitabine. Therefore, we are currently conducting a Phase I dose finding trial of this combination. As a part of this trial, high response rates with excellent tolerance were noted in advanced cutaneous T- cell lymphoma, and are presented here.

METHODS: A 3+ 3 design was used. There were two cohorts of patients: < 65 and ≥65 years of age. The older cohort was dose escalated after the younger cohort demonstrated safety at a dose level. Initial dose escalations were in bortezomib alone, followed by gemcitabine, and then by liposomal doxorubicin.

RESULTS: There were 33 patients who were treated on this trial to date. We are reporting the results of 7 patients with cutaneous T cell lymphoma. These patients had been heavily pretreated with a median of 6 prior therapies (range 1–12). All six of six evaluable patients (100%) achieved a partial remission (PR). Some of these responses are ongoing and the patients are still improving. The most common toxicities were grade 2 thrombocytopenia (50% of patients), grade 1–2 fatigue (44% patients), grade1–2 mucositis (14% patients), and myelosuppression (12% patients). Four patients needed hospitalization early in the course for sepsis. All of these patients had advanced disease with ulcerating skin lesions. Dose limiting toxicity has not been seen.

CONCLUSIONS: The above combination is safe up to the doses tested so far- (dose level 5 = doxil 24mg/m2 on day1, gemcitabine 800 mg/m2 on days 1 and 8 and bortezomib 1.0 mg/m2 on days 1,4,8,11) and has activity in heavily pretreated, advanced cutaneous T-Cell lymphoma.

Responses in Cutaneous T Cell Lymphoma Age Sex Stage Number of prior therapies Dose Level Response Duration of therapy in months 71 F IV 12 1 PR 4 32 F IV 6 1 PR 3 (sent to BMT) 61 F IV 8 2 Lost to follow up after 2 doses 72 M IV 2 2 PR 7+ 73 M IV B 6 2 PR 4+ 53 M II B 1 5 PR 7+ 60 F IV 5 5 PR 8+
Atiprimod Inhibits Growth of Mantle Cell Lymphoma In Vitro and In Vivo and Induces Apoptosis Via Activation of the Mitochondrial Pathway.
Authors: Wang Michael , Zhang Liang , Han Xiaohong , Yang Jin , Qian Jianfei , Wang Siqing , Hong Sungyoul , Zhang Xiang , Zhou Yuhong , Romaguera Jorge , Yi Qing
Published: October 2019

Mantle cell lymphoma (MCL) has poor outcome and is a therapeutic challenge. Atiprimod, a cationic amphiphilic compound, inhibits the proliferation of most human tumor cell lines. Our study was undertaken to evaluate the therapeutic efficacy of Atiprimod on MCL cells and elucidate the mechanism by which it induces cell apoptosis. Four human MCL cell lines, SP53, MINO, Grant 519, and Jeko-1; freshly isolated primary MCL cells from three MCL patients; and normal peripheral blood mononuclear cells (PBMCs) from five healthy donors were treated with Atiprimod. A 3H-thymidine incorporation assay showed that Atiprimod not only inhibited the growth of the MCL cell lines SP53, MINO, Grant 519, and Jeko-1, but also freshly isolated patient MCL cells in a dose-dependent manner. Interestingly, Atiprimod also inhibited the proliferation of PHA-, PMA/ionomycin-, or anti-CD3 mAb/anti-CD28 mAb-activated, but not resting PBMCs from healthy donors. Flow cytometry analysis with fluorescence-labeled Annexin V and propidium iodide showed that Atiprimod induced apoptosis in both the cell lines and primary MCL cells in time-dependent and dose-dependent manners. Notably, Atiprimod did not induce apoptosis of normal T cells and B cells. Atiprimod was also effective and therapeutic in a MCL mouse model established in severe combined immunodeficient (SCID) mice. SP53 cells (5 × 106) were inoculated subcutaneously into the right leg of SCID mice. Three weeks later, after palpable tumors developed, mice were treated intraperitoneally with either vehicle alone (PBS) or Atiprimod (25 mg/kg per day) for 6 consecutive days. Tumor growth was significantly inhibited after Atiprimod treatment compared with vehicle control, and the survival time of tumor-bearing mice was significantly prolonged in the treatment group. Western blot analysis showed that apoptosis of MCL cells was induced through the activation of caspase-9, caspase-3, and PARP pathway. Furthermore, the expression of phosphorylated Bcl-2, Bax, Bad, Bax-xL, and cytochrome C was increased in Atiprimod-treated MCL cells. Pretreatment of cells with caspase-9 inhibitor (z-LEHD) or pan-caspase inhibitor (z-VAD) but not caspase-8 inhibitor (z-IETD) completely blocked Atiprimod-induced apoptosis. In conclusion, Atiprimod inhibits growth and induces apoptosis of MCL cells in vitro and in vivo. Cell apoptosis was induced via activation of the mitochondrial signaling pathway. The therapeutic efficacy of Atiprimod on MCL cells in the mouse model supports the use of Atiprimod as a potential agent in MCL chemotherapy.
Inhibition of the Pan-Bcl-2 Family by the Small Molecule GX15-070 Induces Apoptosis in Mantle Cell Lymphoma (MCL) Cells and Enhances the Activity of Two Proteasome Inhibitors (NPI-0052 and Bortezomib), and Doxorubicin Chemotherapy.
Authors: Yazbeck Victor Y. , Georgakis Georgios V. , Li Yang , McConkey David , Andreeff Michael , Younes Anas
Published: October 2019

Mantle cell lymphoma (MCL) accounts for 6–8% of all non-Hodgkin lymphomas (NHLs). It is an aggressive lymphoma with a poor prognosis--it is generally considered incurable with conventional treatments, and median survival is 3–4 years with a 10-year survival of only 10–15%. There is no accepted standard of care and effective treatments are greatly needed. Bcl-2 family proteins are important regulators of the intrinsic apoptotic pathway and are involved in oncogenesis and chemoresistance of a variety of tumor types, including lymphoma. Antiapoptotic proteins of the Bcl-2 family are overexpressed in mantle cell lymphoma (MCL) cells and may be responsible, in part, for drug resistance. GX15-070 is a small-molecule antagonist of the BH3-binding groove of the Bcl-2 family of proteins, and is currently in Phase I clinical trials. Consequently, we determined the activity of GX15-070 in 3 MCL cell lines (Jeko-1, Mino, and SP53). Cell viability was determined by MTS assay, apoptosis by Annexin-V binding and FACS analysis, and molecular changes by western blot. GX15-070 induced apoptosis in all three MCL cell lines in a dose and time-dependent manner. In the SP53 cell line, GX15-070 decreased MCL-1 and Bak levels, increased Bax and cleaved caspase 3. Furthermore, GX15-070 activated both the extrinsic and intrinsic apoptotic pathway as evident by cleavage of caspase 8, 9, and Bid. Both bortezomib and the novel proteasome inhibitor NPI-0052 induced single agent antiproliferative activity in MCL. GX15-070 enhanced the effect of both proteasome inhibitors. Additionally, GX15-070 showed an additive effect with doxorubicin. These studies suggest that GX15-070 may have a therapeutic value in MCL either alone or in combination with proteasome inhibitors or chemotherapy.