bluuebunny/crossref_metadata_2025 huggingface dataset:
MK-0457, a Novel Multikinase Inhibitor, Is Active in Patients with Chronic Myeloid Leukemia (CML) and Acute Lymphocytic Leukemia (ALL) with the T315I BCR-ABL Resistance Mutation and Patients with Refractory JAK-2 Positive Myeloproliferative Diseases (MPD).
Authors: Giles Francis , Cortes Jorge , Bergstrom Donald A. , Xiao Alan , Jones Dan , Verstovsek Srdan , Thomas Deborah , Kantarjian Hagop M. , Freedman Steven J.
Published: October 2019

MK-0457 (VX-680) is a small molecule inhibitor of aurora kinases A, B, and C, FLT3, and JAK-2 with nanomolar level broad spectrum pre-clinical anti-tumor activity. The T315I BCR-ABL mutation mediates high level resistance to imatinib, dasatinib and nilotinib. MK-0457 has in vitro activity against cells expressing wild-type or mutated BCR-ABL, including the T315I BCR-ABL mutation. A Phase I study of MK-0457 is being conducted in patients with refractory hematologic malignancies. The study regime is a 5-day continuous IV regimen given every 2 to 3 weeks. To date, dose levels of 8 (N=4), 12 (N=5), 16 (N=3), 20 (N=4), 24 (N=15), 28 (N=6), and 32 (N=3) mg/m2/hr have been investigated. No MK-0457-attributable extramedullary Grade 3 adverse events (AEs) have been observed to date, and thus, the maximum-tolerated dose (MTD) has not yet been reached. Grade 1 nausea was observed in 1 patient each at the 12 and 24 mg/m2/hr levels, and there have been 4 patients with Grade 1 alopecia. Dose-proportional PK has been observed across dose levels. Myelosuppression is consistently seen with MK-0457 at all dose levels studied to date and appears dose-related. Of 40 patients on study to date, 15 had CML, 2 had ALL, 13 had acute myeloid leukemia (AML), and 10 had rapidly progressive or transforming MPD. A nested PCR strategy followed by direct DNA sequencing using the dideoxy chain termination method was used to detect and monitor mutations in codons 221 to 500 of the BCR-ABL kinase domain in CML and ALL patients. Of 15 patients with CML, 11 had a T315I BCR-ABL mutation as had 1 of 2 ALL patients. Of 14 currently evaluable patients with CML, 11 had an objective (hematologic, cytogenetic, and/or molecular) response, including all 11 patients with the T315I mutation. The patient with T315I-mutant ALL also had an objective response. Six of 8 currently evaluable patients with JAK-2 positive refractory MPD have achieved an objective response. Downregulation of BCR-ABL and CrkL phosphorylation in leukemia cells from CML and ALL patients treated on study has been documented – the possible role of aurora kinase inhibition in these clinical responses requires further investigation. Accrual to the study is ongoing at the 36 mg/m2/hr dose level. The currently reported cases are the first observed clinical activity of a kinase inhibitor against the T315I positive CML/ALL and JAK-2 positive MPD. The observation of responses in these patients to doses of MK-0457 associated with no significant toxicity warrants further study.
Cancer Stem Cell Targeting in Multiple Myeloma by GRN163L, a Novel and Potent Telomerase Inhibitor.
Authors: Matsui William , Wang Qiuju , Vala Milada , Barber James P. , Meeker Alan , Tressler Robert , Harley Calvin , Huff Carol A. , Smith B. Douglas , Jones Richard J.
Published: October 2019

Telomerase activity (TA) is required within normal cells capable of long-term replication, including stem cells and is upregulated in many cancers. In the absence of TA or presence of TA inhibitors, the progressive shortening of telomeres ultimately results in cellular senescence and/or apoptosis. These observations support that TA inhibitors represent a novel class of anti-tumor agents. Much evidence suggests that human cancers display a hierarchical cellular organization that mirrors normal tissues. Cancer stem cells (CSC) are derived from the malignant transformation of normal stem cells and progenitors and retain the capacity to self-renew. Moreover, CSC give rise to differentiated tumor cells that form the bulk of the tumor mass, but have little or no capacity for long-term proliferation. We recently demonstrated that the malignant CD138+ plasma cells in multiple myeloma (MM) have limited replicative potential; instead they arise from the differentiation of clonogenic CSC that resemble normal memory B cells (CD138negCD19+CD27+). In addition, several groups have demonstrated that telomerase inhibitors are active against human MM cell lines in vitro and in vivo. We examined TA in CD138+ plasma cells and CD138neg precursors, and studied the effects of telomerase inhibition against both cell populations. We isolated CD138+ and CD138neg cells by FACS from three human MM cell lines (RPMI 8225, NCI-H929, and U266) and measured TA using a PCR-based assay of activity. For each cell line, TA was detectable within both the CD138neg and CD138+ cell populations. GRN163L is a lipid conjugated 13 nucleotide thio-phosphoramidate oligonucleotide that acts as a potent and specific active site inhibitor of telomerase. We found that treatment with GRN163L (0.1–5μM) markedly reduced TA within 48 hours. To examine the effects of telomerase inhibition on clonogenic growth, we continuously cultured CD138+ and CD138neg RPMI 8226 cells with GRN163L (1μM). Cells were collected weekly, washed to remove GRN163L, and then plated in methylcellulose to assess colony formation. We found that GRN163L was active against both CD138+ and CD138neg cells and eliminated the colony forming potential of both by 5 weeks. Similarly, we found that GRN163L inhibited the in vitro clonogenic growth of CD138neg MM CSC isolated from the bone marrow aspirates of patients with MM. These data demonstrate that TA is detectable within both immature MM CSC and mature MM plasma cells, and that CSC from both cell lines and primary clinical samples are targeted by the telomerase inhibitor GRN163L. Therefore, this agent may offer a novel therapeutic approach to myeloma as well as other diseases in which CSC have been identified.
Rbm15 Affects Notch Signaling and Myelopoiesis.
Authors: Renda Matthew J. , Cheng Ee-Chun , Wang Lin , Ma Xian-Young , Krause Diane S.
Published: October 2019

Acute Megakaryoblastic Leukemia (AMKL) presents with one of two different genotypic abnormalities; either Down Syndrome (DS) accompanied with GATA-1 mutations, or a non-DS translocation t(1;22)(p13;q13). In AMKL associated with DS, there is trisomy 21 and various partial loss of function mutations in GATA-1. In non-DS AMKL, the translocation t(1;22)(p13;q13) encodes a fusion protein, RBM15-MKL, comprised of the transcriptional co-factors RBM15 (chromosome 1) and MKL (chromosome 22). In order to elucidate the role of the RBM15-MKL fusion protein in AMKL, we must understand the normal functions of RBM15 and MKL. Herein we demonstrate a role for Rbm15 in myeloid differentiation.

Previous work in the lab showed that Rbm15 is expressed at highest levels in hematopoietic stem cells, and at more moderate levels during myelopoiesis of murine cell lines and primary murine cells. Therefore, we hypothesized that Rbm15 plays a role in myeloid differentiation. Indeed, we demonstrate that shRNA-mediated knockdown of Rbm15 enhanced the differentiation of 32DWT18 myeloid precursor cells. Recent studies have shown the Rbm15 homolog, Sharp, interacts with RBPJk, a critical transcription factor in the Notch signaling pathway; and since Notch signaling is essential for hematopoiesis, we hypothesized that Rbm15 may also modulate Notch signaling. We demonstrate that Rbm15 alters Notch-induced HES1 promoter activity in a cell-type specific manner. Rbm15 inhibits Notch-induced HES1 activity in non-hematopoietic cell lines, but stimulates this activity in hematopoietic cell lines including 32DWT18 and HEL (human erythroleukemia). In addition, we show that the N-terminus of Rbm15 co-immunoprecipitates with RBPJk and has a dominant negative effect by impairing the activation ability of HES1 promoter activity by full length-Rbm15.

Thus, Rbm15 is differentially expressed during hematopoiesis and may act to inhibit myeloid differentiation in hematopoietic cells via a mechanism that is mediated, at least in part, by stimulation of Notch signaling via interaction with RBPJk. Consistent with a potential mechanistic role of Rbm15 and Notch in myelopoiesis, we have shown that shRNA-mediated knockdown of Rbm15 in 32DWT18 cells promotes myeloid differentiation, suggesting that the RBM15 component of the RBM15-MKL fusion protein may act by blocking differentiation. Currently, we are examining the effect of RBM15 overexpression and knockdown in primary mouse bone marrow cells using in vitro and in vivo assays.
The Hypomorphic Gata1low Mutation Alters the Proliferation/Differentiation Potential of the Common Megakaryocytic-Erythroid Progenitor.
Authors: Ghinassi Barbara , Sanchez Massimo , Martelli Fabrizio , Amabile Giovanni , Vannucchi Alessandro M. , Migliaccio Giovanni , Orkin Stuart H. , Migliaccio Anna Rita
Published: October 2019

Recent evidence suggests that mutations in the Gata1 gene may alter the proliferation/differentiation potential of hemopoietic progenitors. We had previously described that hematopoietic tissues from mice carrying the hypomorphic Gata1low mutation contain high numbers (~10%) of “unique” progenitor cells that generate colonies composed by erythroblasts, megakaryocytes and mast cells (Migliaccio et al, J Exp Med197:281, 2003). Predicted by the stochastic model of hematopoietic commitment, such a tri-lineage progenitor cell has not been isolated prospectively as yet from the marrow of normal mice. The aim of this study was to clarify the role of Gata1 in hematopoietic commitment by identifying the antigenic profile and proliferation potential of the Gata1low progenitors giving rise to tri-lineage colonies. First, we compared the frequency of granulo-monocytic (GMP), megakaryocytic-erythroid (MEP), common myeloid (CMP) and mast-cell restricted (MCP) progenitors in marrow, and spleen, from wild-type and Gata1low littermates. Next, the different populations were isolated, and their differentiation and proliferation potential characterized under conditions of limiting dilution followed by single cell re-cloning. In Gata1low mice, the frequency of cells with the antigenic profile of CMP, MEP, and GMP was normal in marrow and markedly increased in spleen, while cells with the MCP profile were not detectable. However, mutant cells isolated according to the MEP phenotype, had the “unique” property to generate mast cells and their precursors in 7 days of culture, in addition to erythroblasts and megakaryocytes. Furthermore, the progeny of ~11% of mutant MEP could be propagated in culture, as single cells, with 95% efficiency, for up of 4 months. In comparison, the progeny of wild-type MEP became extinguished in 7–14 days. In agreement with these results, tri-lineage (erythroid, megakaryocytic and mastocytic) cell lines were consistently isolated from bone marrow and spleen cells of Gata1low mice. These results indicate that cells with the phenotype corresponding to myeloid, but not those corresponding to mastocytic, progenitors are detectable in tissues from Gata1low mice. However, in these mice, the mast cell generating activity is abnormally acquired by MEP, that, therefore, are similar in phenotype, but not in function, to wild-type MEP. These results confirm the crucial role played by Gata1 in hematopoietic commitment and identify, as a new target for the Gata1 action, the restriction point at which CMP became either MEP or MCP.
Electroporation of CXCR4 mRNA into Primary Mesenchymal Stem Cells Enables SDF-1 Dependent Migration.
Authors: Ryser Martin F. , Ugarte Fernando , Thieme Sebastian , Gentsch Marcus , Herber Martin , Brenner Sebastian
Published: October 2019

mRNA transfection is a safe alternative to DNA transfection for transient transgene overexpression in mammalian cells as genomic integration of the transferred nucleic acid is impossible. We developed a new mRNA electroporation protocol for transfection of primary mesenchymal stem cells (MSCs) using a hypoosmolar buffer system. Polyadenylated and capped mRNA was in vitro transcribed from DNA templates by T7 RNA polymerase. The DNA template generation has been simplified by our group using PCR amplification of the transgene sequence with primers providing the T7 RNA polymerase promoter and optimized 5′ and 3′ UTR sequences for better translation and RNA stability. This manoever allows to generate the DNA template from any plasmid vector and bypasses the need for additonal cloning steps. Canine primary MSCs were isolated by selection of adherent cells from bone marrow aspirates in a low glucose medium. Transfection of GFP or CXCR4 mRNA into primary MSCs resulted in transgene overexpression in more than 90% of target cells with excellent viabilities (>95%). Expression of CXCR4 was well detectable for up to 96 hours after transfection. SDF-1 dependent Ca-flux was only present in CXCR4-transgene overexpressing MSCs suggesting functional expression of the chemokine receptor. Transwell migration experiments demonstrated a 2 fold increased migration of CXCR4 mRNA transfected MSCs towards a gradient of SDF-1 compared to background migration without SDF-1. GFP mRNA transfection did not result in any SDF-1 stimulated migration. Our data demonstrate efficient and safe transient genetic engineering of primary MSCs based on mRNA transfection. mRNA transfection induced chemotaxis will facilitate receptor mediated targeting of transplanted MSCs at specific body compartments.
A Novel Peptide Containing a Nine Amino Acid Sequence from Nur77 Induces Bcl-2-Dependent Apoptosis in AML.
Authors: Andreeff Michael , Konopleva Marina , Watt Julie C. , Samudio Ismael J. , Satterthwait Arnold C. , Kitada Shinichi , Zhang Xiao-kun , Reed John C.
Published: October 2019

Nur77 (also known as TR3 and NGFI-B) is a nuclear orphan receptor that in response to various stimuli can translocate from the nucleus to the mitochondria, bind Bcl-2, and induce a conformational change in Bcl-2 that exposes its BH3 domain. This conformational change of Bcl-2 transforms this protein into a pro-apoptotic molecule that can induce cytochrome c release and apoptosis (Cell 116:527, 2004). Interestingly, in acute myeloid leukemia (AML) cell lines and blasts, Nur77 is absent. We have recently generated a nine amino acid peptide from the Nur77 protein (TR3) that is capable of inducing the same pro-apoptotic conformational change in Bcl-2 that Nur77 elicits (manuscript submitted). We tested the hypothesis that a cell-permeable version of this peptide can induce Bcl-2-dependent apoptosis in AML cells and AML stem cells. Primary AML samples (n=6) were incubated with 20 μM control or TR3 peptide for 24 hrs. Induction of apoptosis was measured as CD34+38−123+ PS/Annexin V+ by multiparametric flow cytometry. Our results demonstrate that the L-enantiomer form of TR3 indeed induces apoptosis in total AML cells (60%, p=0.001) and CD34+38−123+ stem cells (67%, p=0.001), as compared to control peptide. Bax expression did not affect the peptide’s ability to induce apoptosis, as wild-type HCT116 cells and HCT116 cells deficient in Bax showed similar levels of apoptosis after treatment with the TR3 peptide. However, Bcl-2 expression was shown to be critical since lentiviral shRNA ablation of Bcl-2 in KG1 cells completely prevented induction of apoptosis by TR3 peptides. We conclude that the TR3 peptide is a potent inducer of apoptosis that mimics the action of Nur77 on Bcl-2 at the mitochondrial level. These results suggest that the Nur77 peptide or compounds that mimic this peptide may have utility as a novel therapeutic agent against Bcl-2 expressing cancers and leukemias.
PDE4 Inhibitors Augment Glucocorticoid Receptor Levels in B-CLL Cells but Not in T Cells, B Cells, Monocytes or Neutrophils.
Authors: Lerner Adam , Meyers John A. , Taverna Josephine A. , Makkinje Anthony
Published: October 2019

cAMP analogues have long been known to induce apoptosis in specific lymphoid subsets and to augment the apoptotic effect of glucocorticoids. However, a clinically feasible means by which to take advantage of these effects for the treatment of lymphoid malignancies has not previously been identified. Inhibition of type 4 cAMP phosphodiesterases (PDE4) activates cAMP-mediated signaling in many cell types and several PDE4 inhibitors are currently in clinical trials for inflammatory illnesses such as asthma, COPD and psoriasis. We have previously reported that PDE4 inhibitors induce apoptosis in B-CLL and augment the apoptotic effects of glucocorticoids such as dexamethasone. However, the mechanism by which PDE4 inhibitors increase glucocorticoid-induced apoptosis has remained unclear. In gene chip studies, we find that the PDE4 inhibitor rolipram (10 uM) augments transcript levels of glucocorticoid receptor alpha (GRa). A rolipram-induced dose and time-dependent increase in GRa levels was confirmed both by real-time PCR and by Western analysis. In leukemic cells derived from eight B-CLL patients, GRa transcript levels rose over the first four hours to a mean of 4.8 +/− 0.2-fold above baseline and maintained such a fourfold increase for at least 24 hours. An analysis of GRa transcript half-life following actinomycin D treatment demonstrates that rolipram augments transcript levels in B-CLL cells by a transcriptional mechanism. Consistent with this, rolipram treatment augments GRa transcripts derived from promoters 1A and 1C to a greater degree than 1B. Remarkably, rolipram does not augment GRa transcript levels in normal T cells, B cells, monocytes or neutrophils, even in the presence of the adenylate cyclase activator forskolin. Similarly, no increase in GRa transcript levels was observed in leukemic T-CLL cells. As previously reported in B cell lines, treatment of B-CLL cells with dexamethasone reduced GRa transcript levels in a dose-dependent manner. Co-treatment with PDE4 inhibitors maintained GRa transcript levels above basal levels even at the highest dexamethasone concentration tested (1 uM). Finally, we have confirmed the ability of PDE4 inhibitors to augment dexamethasone-induced apoptosis using several PDE4 inhibitors currently in clinical trials for other indications. Our results suggest that co-treatment with PDE4 inhibitors and high-dose dexamethasone may increase leukemic cell GRa levels and augment the apoptotic efficacy of glucocorticoid therapy in B-CLL patients.
Clinical Features and Prognosis of Patients with Myelodysplastic/Myeloproliferative Syndrome-Unclassified (MDS/MPD-U): Refractory Anemia with Ringed Sideroblasts with Thrombocytosis (RARS-T) Is a Favorable Prognostic Subgroup.
Authors: Atallah Ehab , Kantarjian Hagop , Cortes Jorge , Pierce Sherry , Estey Elihu , Garcia-Manero Guillermo , Thomas Deborah , Ravandi-Kashani Farhad , Giles Francis , Verstovsek Srdan
Published: October 2019

MDS/MPD-U is a newly defined clinical entity in the WHO classification. It includes patients (pts) with features of both myelodysplasia and myeloproliferation that cannot be classified under any other WHO category. In addition it includes the provisional entity RARS-T. Recent reports found an increased incidence of JAK-2 mutation in pts with RARS-T, suggesting possibly a different biological behavior. The clinical outcome of MDS/MPD-U, and in particular RARS-T, pts have not been well described.

We reviewed 142 patients in the MD Anderson Leukemia Department patient database with the diagnosis of MDS/MPD or MPD-U seen from September 1987 to July 2006, and identified 32 pts that fulfilled MDS/MPD-U criteria according to WHO classification. The median age was 64 years (range 21–82), with a male: female ratio of 3:1. Six pts had RARS-T (19%). Six pts had a previous malignancy (19%), of which 3 received radiation therapy; no chemotherapy was given. Twenty-five pts (73%) were symptomatic at presentation, including fatigue in 17 (53%), weight loss in 13 (41%), fever in 2 (6%), and night sweats in 7 (22%) pts. Fifteen (45%) pts were transfusion dependent. The median hemoglobin was 9.6 g/dl (range 4.8–15.3), white blood cell count 9x109/L (range 0.6–141), platelets 234x109/L (range 7–1687), and percentage of bone marrow blasts 3% (range 0–16). Fifteen pts (45%) had more than one lineage dysplasia and 22 pts (69%) had increased reticulin in the bone marrow. Cytogenetic analysis revealed diploid cytogenetics in 13, trisomy 8 (either alone or with other abnormalities) in 8, complex cytogenetics in 3, and others in 8 pts. Twenty-two pts were treated with multiple different therapies, including hypomethylating agent in 8, with no sustained responses to any therapy. Six pts received supportive care only and 4 are unknown. Two pts underwent bone marrow transplant; one is in remission for >4 years and the other died from acute graft versus host disease. Median follow up time for alive pts was 10.6 months (range 0.3–147). Median survival of the whole group was 19 months (95% CI; range 8–29). Of the 6 pts with RARS-T, 5 had diploid cytogenetics, two had prior malignancy and 5 were transfusion dependent at the time of diagnosis. Patients with RARS-T had a significantly better survival than the other pts (median survival not reached vs. 12 months; p=0.019; Figure 1).

In conclusion, pts with MDS/MPD-U have a poor prognosis with a median survival of one year. However, RARS-T represents a subgroup of pts with better prognosis and long overall survival justifying their separate categorization.

Overall Survival Overall Survival
Both the Endoplasmic Reticulum and the Mitochondria Are Involved in Apoptosis of Erythroid Precursors in Low Grade Myelodysplastic Syndromes.
Authors: Gyan Emmanuel , Frisan Emilie , Deschemin Jean-Christophe , Randriamamptita Clotilde , Dubart-Kupperschmitt Anne , Beyne-Rauzy Odile , Dreyfus Francois , Guesnu Martine , Lacombe Catherine , Mayeux Patrick , Solary Eric , Fontenay Michaela
Published: October 2019

Cell death by apoptosis accounts for the ineffective erythropoiesis that characterizes low grade myelodysplastic syndromes (LG-MDS). We have shown that the death receptor Fas was over-expressed at the surface of LG-MDS erythroid precursors. In an ex vivo culture of bone-marrow CD34+ cells undergoing differentiation into red cells, we observed that apoptosis of LG-MDS erythroid precursors resulted from Fas-mediated activation of caspase-8. Both a Fas-Fc construct and the ectopic expression of a dominant negative mutant of the adaptor FADD (Fas-Associated Death Domain) could prevent the death of MDS erythroid precursors.

In this ex vivo culture system, apoptosis of erythroid precursors was associated with the release of cytochrome c from the mitochondria to the cytosol. To further address the mitochondria involvement in this apoptotic process, we ectopically expressed, by the use of a lentiviral vector, the anti-apoptotic protein Bcl-2 in MDS (n=20) and normal (n=10) CD34+ cells before inducing their erythroid differentiation. Bcl-2 expression delayed the erythroid differentiation of MDS as well as normal CD34+ cells. It also prevented phosphatidylserine exposure, mitochondrial membrane permeabilization, cytochrome c release, caspase-9 and -3 activation in LG-MDS erythroid precursors. Together with the cleavage of Bid, specifically observed in LG-MDS samples, these observations indicated that the Fas-mediated pathway that led to apoptosis of LG-MDS erythroid precursors was connected to the mitochondrial pathway to death.

We also addressed the role of the endoplasmic reticulum (ER) in this death pathway by using a lentiviral vector encoding an ER-targeted Bcl-2 construct, flag-Bcl2-Cb5. The corresponding protein co-localized with the ER protein calreticulin. ER-targeted Bcl-2 prevented apoptosis induced by thapsigargin, an inhibitor of endoplasmic/sarcoplasmic ATPases (SERCAs), without inhibiting apoptosis induced by lonidamine, a mitochondria targeting agent. Like wild-type Bcl-2, ER-targeted Bcl-2 inhibited phosphatidylserine exposure, mitochondrial membrane permeabilization, cytochrome c release, caspase-9 and caspase-3 activation that characterize spontaneous apoptosis of LG-MDS erythroid precursors in culture. Although ER calcium stores were increased in LG-MDS erythroid precursors, the release of calcium was not modulated by ER-targeted Bcl-2 in these cells. Altogether, our results indicate that, in LG-MDS erythroid precursors, spontaneous apoptosis starts at level of the death receptor Fas, then connects to the mitochondrial pathway that functions downstream of the ER through a calcium-independent and Bcl-2-regulated pathway.
Abrogation of the DNA-Damage Response in De Novo AML Versus MDS Cell Lines.
Authors: Boehrer Simone , Ades Lionel , Fabre Claire , Fenaux Pierre , Kroemer Guido
Published: October 2019

Background: The increasing evidence that activation of the DNA-damage-response (DDR) pathway in preneoplastic lesions of solid neoplasms hinders tumorigenesis prompted us to evaluate the same hypothesis in cell line models representing different subtypes of MDS or AML.

Methods: myeloid cell lines corresponding to different subgroups of MDS, having or not progressed to AML (p39, MOLM-13) and de novo AML (MV4-11, and KG-1) were assessed for their capacity to induce a DDR. Using the quintessential inducer of a DDR, the cell lines were gamma-irradiated (2, 5 and 10Gy) and their ability to arrest the cell cycle in G2/M and to undergo apoptosis were assessed.

Results: in contrast to the de novo AML-derived KG-1 cells - the irradiated MDS/AML-derived cell lines P39 and MOLM-13 exhibited an earlier and more pronounced arrest of the cell cycle in G2/M, as well as a higher percentage of apoptotic cells. Whereas 10 Gy induced a G2/M-arrest in about 30% of KG-1 cells, this percentage increased to about 60% in P39 and MOLM-13 cells. Comparably, whereas KG-1 cells exhibited about 15% of apoptotic cells following irradiation with 10Gy, the percentage reached 30% in P39 and MOLM-13 cells. Assessing the underlying molecular determinants we demonstrated that irradiated P39 and MOLM-13 cells retained the ability to activate crucial mediators of the DDR, whereas this ability was decreased in the de novo AML cell line KG-1. In particular, in comparison to KG-1 cells, irradiated P39 and MOLM-13 cells exhibited a more pronounced upregulation of the crucial cell cycle/checkpoint regulators Chk1-P-Ser317 and Cdk1-P-Tyr15. Noteworthy, MDS cell lines with an increased G2/M-arrest exhibited a higher expression of nucleophosmin - which serves as a shuttling protein between the nucleus and the cytoplasm - and were able to retain this increased expression upon irradiation. Moreover, elevated expression of NPM in MDS cell lines corresponded to higher PCNA-expression, which serves as an essential component of the DNA-repair machinery. To further investigate the functionality of the DDR-pathway, the different cell lines were gamma-irradiated in the presence of the ATM-inhibitor KU-55933 and the Chk1 inhibitor UCN-01. Whereas inhibition of Chk1 (and to a lesser extent inhibition of ATM) was able to hinder G2/M-progression in irradiated MDS cells, it remained without effect in the AML cell lines KG-1 and MV4-11. Noteworthy, whereas irradiated P39/MDS cells hindered to activate Chk1 decrease expression of Polo-like-kinase-1 (Plk-1) - which not only governs mitotic entry and exit, but also functions as an oncogene - KG-1 cells maintained a high expression of Plk-1, comparable to that observed in non-irradiated cells.

Conclusion: we demonstrated the different capacities of MDS (or AML post MDS) and de novo AML cell lines to activate the DDR and thus provide evidence that the DDR-pathway is abrogated in de novo AML as compared to MDS cells.
Clofarabine Is Active in Myelodysplastic Syndrome (MDS).
Authors: Faderl Stefan , Gandhi Varsha , O’Brien Susan , Giles Francis , Cortes Jorge , Plunkett William , Kantarjian Hagop M.
Published: October 2019

Background. Clofarabine, an adenosine nucleoside analog, is active in acute myeloid leukemia (AML) and acute lymphoid leukemia (ALL). Its anti-MDS activity is not well defined.

Study Aims. To evaluate the safety and efficacy of clofarabine in MDS in two studies, one with intravenous administration and one with oral, using lower doses than those for leukemia (40–52 mg/m2 IV daily x 5).

Study Group and Results. The first study randomized patients in a Bayesian design to receive clofarabine 15 or 30 mg/m2 IV daily x 5 every 4–6 weeks. The second study used clofarabine 40 mg/m2 orally daily x 5 every 4–6 weeks (assuming oral bioavailability about 50%). Standard eligibility criteria for MDS were used; only high risk MDS patients (IPSS intermediate or high) were eligible. So far, 16 patients have been treated, 10 on IV clofarabine and 6 on oral clofarabine. Age ≥60 yr in 13 (81%); prior therapy with decitabine/azacitidine 11 (69%); cytogenetic abnormalities 12 (75%). Responses were graded according to International Working Group criteria (IWG). Results are shown in the table below.

Myelosuppression was significant resulting in febrile episodes and hospitalization in 4/6 patients on oral clofarabine and in 4/10 patients on IV clofarabine. Rash grade 3 was noted in 1 patient on oral clofarabine.

Summary. Clofarabine is active in MDS. The oral dose of 40 mg/m2 daily x 5 may be too high for MDS; lower dose of oral clofarabine 30 mg/m2 orally daily x 5 will be tested with PK studies. The 2 IV schedules (15 vs. 30 mg/m2) are ongoing.

Oral Clofarabine IV Clofarabine • Treated/evaluable to date 6/3 10/6 • Response - CR 2 (67%) 3 (50%) - Other IWG response -- 1 (17%)
Efficacy of Decitabine in the Treatment of Patients with Chronic Myelomonocytic Leukemia (CMML).
Authors: Wijermans P.W. , Lübbert Michael , Baer Maria R. , Slack James L.
Published: October 2019

CMML is a preleukemia that results in cytopenias, dysplastic morphology of bone marrow and peripheral blood cells, produces large numbers of ineffective monocytes, and is notoriously hard to treat. The recent revision of the WHO classification excludes CMML from the myelodysplastic syndromes (MDS), and reclassifies it as a myelodysplastic/myeloproliferative disease. Decitabine (Dacogen™) is a cytosine analog that reverses aberrant DNA hypermethylation, leading to re-expression of silenced tumor suppressor genes. The reclassification of CMML has led to a review of CMML patients treated with decitabine. Overall response rates (ORR) (complete response [CR] + partial response [PR]) from one pivotal phase 3 trial (D-0007) and two phase 2 trials (PCH 95-11, PCH 97-19) in the subset of patients with CMML receiving decitabine were reviewed. The two phase 2 trials were open-label and single-arm, with a recommended minimum of 4 treatment cycles and a maximum of 8 cycles. The phase 3 trial used a 1:1 randomized comparison of decitabine plus supportive care (SC) vs SC alone with a maximum of 10 cycles of therapy. The phase 3 trial study design dictated that patients be removed from therapy following 8 cycles of decitabine if CR was not achieved, and following 6 cycles in the absence of PR. Patients who maintained a CR for 2 cycles were removed from therapy. For consistency across trials, all decitabine-treated patients were evaluated using the phase 2 response criteria (CR was defined by normocellular bone marrow with <5% blasts, and normal Hgb, WBC, and platelet counts, and PR required 50% decrease in blast count, increases in Hgb by >1.5 mmol/L, WBC count by >1000, and platelet count by >50,000). A total of 28 patients diagnosed with CMML are included in this review. Similar demographics and disease characteristics were observed in all 3 studies, with an average age of 70.2 years and 71% of patients male. A baseline WBC of >20,000 was observed in 8/28 (29%) patients and baseline bone marrow blasts >5% were observed in 11/28 (39%) patients. All clinical responses were centrally reviewed. The ORR was 25% (14% CR + 11% PR). Hematologic improvement was observed in 11% of patients and stable disease in 39% of patients. The decitabine adverse event profile seen in CMML patients was similar to observations in other hematologic patient populations, with myelosuppression and related infectious complications. These data demonstrate encouraging activity for decitabine in CMML, and suggest that studies in other myeloproliferative diseases may be warranted.
Long-Term Survival of Patients with Myelofibrosis Treated with Allogeneic Hematopoietic Stem Cell Transplantation.
Authors: Donato Michele L. , Pecora Andrew L. , Goldberg STuart L. , Hsu Jack W. , Siegel David S. , Goy Andre H. , Rowley Scott D.
Published: October 2019

Myelofibrosis with myeloid metaplasia (MMM) is a disorder characterized by clonal myeloproliferation, ineffective erythropoiesis, extramedullary hematopoiesis and bone marrow fibrosis. Between 4/1998 and 1/2006, 17 patients with MMM were treated with allogeneic hematopoietic stem cell transplantation. Patients included 8 males and 9 females with a median age of 51.8 years (range 37–66). All patients were transfusion-dependent at the time of transplantation, 3 (18%) had abnormal cytogenetics and 4 (24%) had a prior splenectomy. Six patients (35%) received a reduced intensity conditioning regimen consisting of either fludarabine and busulfan (1 pt), fludarabine and low dose total body irradiation (1 pt), fludarabine and melphalan (3 pts) or fludarabine and cyclophosphamide (1 pt). Eleven patients (65%) received an ablative regimen of either busulfan and cyclophosphamide (10 pts) or cyclophosphamide and total body irradiation (1 pt). Patients with unrelated donors also received ATG as part of their conditioning. The hematopoietic cell source was peripheral blood in 12 pts (71%) and bone marrow in 5 pts (29%). Six pts (35%) received cells from an HLA-identical sibling, 1 pt (6%) had a 1 Antigen mismatch sibling and 10 pts (59%) received stem cells from an unrelated donor. Graft-versus-host disease prophylaxis consisted of tacrolimus in 15 pts (88%) and cyclosporine in 2 pts (12%).

Transplant-related mortality was 17% (sepsis 1 pt, VOD 1 pt, acute GVHD 1 pt) all occurring in the group treated with a myeloablative regimen. Of the remaining 14 patients, all achieved hematological recovery and transfusion independence. There were 2 relapses and one patient died of disease recurrence; both relapses occurred in the group receiving a reduced intensity regimen. The other 12 patients remain alive and in remission. The 6-year actuarial survival is 67% with no statistical difference between the groups who received a reduced intensity versus a myeloablative regimen.

In conclusion, Myelofibrosis with Myeloid Metaplasia can be successfully treated with allogeneic hematopoietic stem cell transplantation with a significant proportion of patients achieving long-term survival. Hematopoietic recovery can be achieved despite the presence of marrow fibrosis. The optimal preparative regimen remains to be determined with more relapses observed in the reduced intensity regimen group and a higher mortality seen in the myeloablative group. A risk-stratification strategy for regimen selection should be considered in future studies.
A Phase I Open Label Dose Escalation Study To Evaluate MEDI-507 in Patients with CD2-Positive T-Cell Lymphoma/Leukemia.
Authors: Casale Deborah A. , Bartlett Nancy L. , Hurd David D. , Foss Francine , Pro Barbara , Sokol Lubomir , Lieberman Ronald , Luz Hammershaimb
Published: October 2019

This ongoing multicenter study is a Phase I trial with MEDI-507 (Siplizumab) [a humanized IgG1k class monoclonal antibody that binds to the CD2 receptor on human T- and NK-cells] to determine the maximum tolerated dose (MTD) or the optimum biologic dose (OBD) in patients with relapsed/refractory CD2-positive T-cell lymphoma/leukemia [CD2-positive adult T-cell leukemia (ATL), cutaneous T-cell lymphoma (CTCL), peripheral T-cell lymphoma (PTCL), and large granular lymphocytic leukemia (LGL).] Open label 3+3 dose escalation was conducted in which patients receive bi-weekly infusions of MEDI-507 over 3 consecutive days at total doses of 0.7 mg/kg, 3.4 mg/kg or 4.8 mg/kg. Predose and serial MEDI-507 pharmacokinetics (ELISA) at Visit 2 and anti MEDI-507 antibodies (ELISA), peripheral blood total T-Cell and CD2-positive T-cells (flow cytometry), and C3 and C4 complement are obtained for each patient. Patients are followed for one year after their last dose of MEDI-507 for tumor assessment and CD2-positive T-cell recovery. 16 patients have been enrolled: 3 (0.7 mg/kg); 9 (3.4); 4 (4.8). Three additional patients were added to the 3.4 mg/kg cohort to replace patients who progressed early and one patient with tumor lysis syndrome. Diagnoses of the 16 patients: PTCL (9), CTCL (6) and NK-LGL (1). Frequent adverse events reported, to date, are infusion reaction (7/16) patients; hypertension (4/16); lymphopenia (4/16); fatigue (4/16) and leukopenia (3/16). Two DLT’s have been observed. The first, erythematous confluent dermatitis, occurred in one patient at 3.4 mg/kg and the cohort was expanded with eventual dose escalation. The second, pulmonary edema, occurred in one patient at 4.8 mg/kg and the cohort is currently being expanded without further DLT identified to date. Two responses have been observed, one PR in an NK-LGL patient at 3.4 mg/kg and one CR in a PTCL patient at 3.4 mg/kg. MEDI-507 has been well tolerated and shown anti-tumor activity. Dosing will change to weekly dosing with dose escalation (3+3) starting at 1.2 mg/kg as 0.8 mg/kg was seen to be safe in the single center study. MTD and/or OBD have not been determined.
RHAMM/CD168 Is a Novel Leukemia Associated Antigen with Prognostic Value for Patients with B-Cell Chronic Lymphocytic Leukemia.
Authors: Giannopoulos Krzysztof , Krober Alexander , Dmoszynska Anna , Rolinski Jacek , Dohner Hartmut , Stilgenbauer Stephan , Schmitt Michael
Published: October 2019

Background and Aims: Differential expression of molecules in patients with B-cell chronic lymphocytic leukemia (B-CLL) might define suitable targets for T cell based vaccines and/or antibody approaches.

Methods: We assessed the mRNA expression of the tumor associated antigen (TAA) RHAMM/CD168 defined earlier by serological analysis of cDNA expression libraries (SEREX) from leukemic cells.

Results: Peripheral blood mononuclear cells from 40 B-CLL patients and 20 healthy volunteers (HVs) were examined by quantitative RT-PCR. A leukemia-restricted expression of the antigen RHAMM/CD168 was observed in 39/40 B-CLL patients in contrast to the absence of its expression in HVs. To evaluate the immunogenicity of this novel LAA, mixed lymphocyte peptide cultures (MLPCs), followed by enzyme-linked immunosorbent spot (ELISPOT) and flow cytometry assays were performed to detect antigen-specific CD8+ T cells. RHAMM/CD168 specific responses by CD8+ HLA-A2/R3tetramer+CCR7-CD45RAhigh effector T cells were detected. As these CD8+ T cells contribute to the elimination of RHAMM+ CLL cells, we questioned whether expression of the antigen would be associated with a better survival. RHAMM/CD168 expression revealed to be higher in patients with unmutated IgVH status. RHAMM normalized against the housekeeping gene TATA binding protein (TBP), i.e. the RHAMM/TBP ratio was defined as a prognostic surrogate marker for B-CLL. B-CLL patients with a RHAMM/TBP ratio > 1.67 showed a significantly shorter treatment free survival (TFS) (Fig. 1). A tendency towards higher RHAMM/TBP expression ratios was observed in B-CLL cases with del11q.

Conclusion: RHAMM/CD168 is a novel LAA in B-CLL patients, an antigen correlating with the clinical course of the disease. Therefore, we consider RHAMM/CD168 an interesting target for immunotherapy in early stage B-CLL patients, especially with worse prognosis (IgVH unmutated).

Figure 1. Treatment-free-survival (TFS) according to RHAMM/TBP ratio Figure 1. Treatment-free-survival (TFS) according to RHAMM/TBP ratio
Integrating Prognostic Markers and Cellular Signaling Identifies More Chronic Lymphocytic Leukemia Patients with Adverse Prognosis.
Authors: Brennan Paul , Alghazal Suhair , Lloyd Naomi Price , Williams Janet E. , Griffiths Jeff D. , Rowntree Clare , Pratt Guy , Fegan Chris , Pepper Chris
Published: October 2019

In this study we devised an equation that provides a new way of identifying B-cell chronic lymphocytic leukemia (CLL) patients who will require early therapeutic intervention. The equation was developed by studying the biology of the disease with a focus on cell signal transduction. Using statistical analysis, we show that measured tyrosine phosphorylation can be described as a function of VH gene mutation status, and the expression of ZAP-70 and CD38 (r2 = 0.81, n = 49). Furthermore, we show that tyrosine phosphorylation has biological relevance as it sets a threshold for the activation of the transcription factor, NF-kB, resulting in the modulation of cell survival. Using the equation derived from our training dataset we calculated tyrosine phosphorylation in a cohort of 155 unselected CLL patient samples. The patient cohort was then divided into those with calculated tyrosine phosphorylation above or below the threshold required to activate NF-kB. Patients with high calculated tyrosine phosphorylation had a shorter time to first treatment. This method identified more patients at risk of requiring early treatment than VH gene mutation status, ZAP-70 or CD38. Furthermore, calculated tyrosine phosphorylation identifies patients that will require early intervention within the mutated VH gene cohort and even patients that have Stage A disease.
Expression of MicroRNA (miR) miR-15a/miR-16-1 Downregulates Expression of BCL-2 Protein in Chronic Lymphocytic Leukemia.
Authors: Chen Liguang , Tang Li , Calin George , Croce Carlo M. , Kipps Thomas J.
Published: October 2019

MicroRNAs (miRNAs) comprise a family of small RNA, each member of which can potentially regulate post-transcription gene expression in a temporal and tissue-specific manner. Each miRNA encodes a transcript of about 22 nucleotides that can modulate expression of specific target mRNA. We identified that the chronic lymphocytic leukemia (CLL) cells of a large proportion of patients have aberrant, low-level expression of two clustered miRNAs located at 13q14, designated miR-15a and miR-16–1, providing the first example of dysregulated miRNA expression in a human cancer (PNAS USA99:15524, 2002).

These miRNAs each have sequences that potentially could target mRNA encoding the anti-apoptotic protein bcl-2, which is expressed at high levels in CLL. Transfection of a lymphoma B cell line lacking expression of miR-15a and miR-16–1 with an expression vector encoding miR-15a and miR-16–1, designated pmiR-15/16, attenuated the expression-level of bcl-2 protein and enhanced the susceptibility of such lymphoma B cells to apoptosis in vitro. However, it was not certain whether miR-15a and miR-16–1 could modulate expression of bcl-2 in primary leukemia cells that were not adapted for propagation in vitro. We selected primary CLL cell samples (n = 3) that harbored deletions at 13q14 and that lacked expression of miR-15a and miR-16–1, as assessed via microRNA array and quantitative RT-PCR analyses. We transfected these cells with pmiR-15/16 or a control vector via electroporation and, in parallel studies, also transfected these CLL cells with sense and antisense control oligo-RNAs via transmessenger transfection. Before and after such manipulations we monitored for expression of miR-15a and miR-16–1 by RT-PCR, for relative expression of BCL-2-family member transcripts using a multiplex ligation-dependent probe amplification (MLPA) assay, and for expression of bcl-2 protein using immunoblot analysis and flow cytometry. We found that CLL cells transfected with either pmiR15/16 or with miR-15a and miR-16–1 sense oligo-RNAs had increased expression-levels of miR-15a and miR-16–1 within 24 hours after transfection, whereas CLL transfected with the control vector or antisense oligo-RNA did not. Depite high-level expression of miR-15a and miR-16–1, the relative levels of BCL-2 transcript did not change over the 48 hours after transfection that we examined. However, in this time period we observed that CLL cells made to express miR-15a and miR-16–1, experienced significant reductions in the levels of bcl-2 protein, which were not observed in control transfected CLL cells. This is the first demonstration that miRNAs can effect post-transcriptional regulation of protein expression in a primary human tumor and suggest that miRNAs may have potential therapeutic utility in the modulation of pathogenic gene-expression in CLL and other cancers.
Activation of NFAT and NFkB Transcription Factors upon B Cell Receptor (BCR) Stimulation Is Restricted to Evolutive Chronic Lymphocytic Leukemia (CLL) Cases.
Authors: Chevallier Nathalie S. , Deglesne Pierre-Antoine , Beitar Taoufik , Baran-Marszak Fanny , Letestu Remi , Vlad Amalia , Lefebvre Valerie , Clavereau Isabelle , Cymbalista Florence , Varin-Blank Nadine
Published: October 2019

Chronic lymphocytic leukemia (CLL) is a highly heterogeneous disease in which BCR signaling capacity plays a central role in disease progression. Previously, we have shown that BCR engagement allows the identification of two groups of patients with a strong correlation between the in vitro cell survival response and the biological criteria (IgVH somatic hypermutations, ZAP 70 expression ) or the clinical stage. One group, corresponding to non evolutive patients, showed absence of a substantial BCR signaling (Non responder). In contrast, BCR signaling promoted in vitro cell survival in the group of Responder cases corresponding to evolutive patients. In the latter only, response to BCR ligation was associated with induction of NFkB and NFAT regulated genes, such as Cyclin D2 and CD23.

Since NFAT and NFkB transcription factors, regardless of the patients clinical status, have been described as constitutively nuclear in B-CLL cells, the aim of our study was to investigate whether the functional implication of these factors was different between our two groups. First, we confirmed by confocal microscopy and band shift analysis that localization of NFAT and NFkB was uniformely nuclear independently of the CLL status and the BCR response capacity. By RQ-PCR , we also investigated the expression of the various isoforms of NFAT (NFAT1, 2, 3 and 4) and showed that their expression profile was similar to normal B cells. Despite the nuclear localization of NFkB and NFAT, DNA binding capacity was only enhanced in cases responsive to BCR stimulation. Finally, using specific Tat-peptide inhibitors of NFAT and NFkB nuclear transport upregulation of cyclin D2 and CD23 was abrogated and survival advantage was lost in responder cases thereby confirming the crucial role of these factors upon BCR stimulation. However, in non responder cases in which no CD23 or cyclin D2 induction was present, this inhibition had no impact on cell survival upon BCR stimulation . Nonetheless, these cells retained the ability to respond to ionomycin or PMA reflecting functional PKC/calcium-calcineurin signaling pathways. Altogether, our results showed that NFAT and NFkB were activated in responder cells only despite their homogenous expression in all cases. In conclusion, the inability of the cells from stable B-CLL cases to activate the NFAT and NFkB pathways upon BCR signaling is not related to a functional defect or abnormal expression of these factors but likely reflects an early signaling defect. Moreover, our results show that the use of peptide inhibitors may restore a stable profile in B-CLL cells from evolutive patients.