bluuebunny/crossref_metadata_2025 huggingface dataset:
A Phase I/II Trial of the Kinesin Spindle Protein (KSP) Inhibitor SB- 743921 Administered on Days 1 and 15 Every 28 Days without and with Prophylactic G-CSF in Non-Hodgkin or Hodgkin Lymphoma.
Authors: Gerecitano John , O’Connor O. , Van Deventer H. , Hainsworth J. , Leonard J. , Goy A. , Afanasyev B. , Chen M. , Saikali K. , Conlan M. G , Escandon R , Wolff A
Published: April 2020

Background: KSP is a mitotic kinesin essential for cell cycle progression. SB-743921 (SB-921), a selective KSP inhibitor, blocks mitotic spindle assembly with cell cycle arrest in mitosis and cell death. Since neurons lack a mitotic spindle, neurotoxicty, common with anti-tubulins, is not expected. In the first-in-humans (FIH) trial, the maximum tolerated dose (MTD) was 4 mg/m2 q21 days (d), a dose density of 0.2 mg/m2/d. Since neutropenia was the major dose-limiting toxicity (DLT), with nadir at ~d8 and recovery by ~d15, a q14d schedule without (−GCSF) and with prophylactic G-CSF (+GCSF) is being explored in this trial.

Methods: In Phase I of this Phase I/II trial, the MTD of SB-921 (−GCSF) and (+GCSF) will be determined. In Phase II, efficacy and safety of the MTD will be further explored. Eligible patients (pts) have relapsed or refractory Hodgkin (HL) or non-Hodgkin (NHL) lymphoma, aggressive (a) or indolent (i), have had at least 1 prior chemotherapy (CT) regimen, and have relapsed after or were not candidates for autologous stem cell transplant. SB-921 is given to cohorts of 3 on d1/d15 q28d, starting at 2 mg/m2 and escalating by 1 mg/m2. Cohorts expand to 6 if 1/3 pts have DLT. Once DLT (−GCSF) is identified, (+GCSF) dosing begins at the (−GCSF) MTD, escalating in 1 mg/m2 increments until (+GCSF) DLT is identified.

Results: The (−GCSF) cohort included 39 pts treated at 6 dose levels (2–7 mg/m2) of SB-921. DLT was reported in 4 pts: 2/10 at 6 mg/m2 (both neutropenia with sepsis) and 2/7 at 7 mg/m2 (both Grade 4 neutropenia lasting >5d). MTD (−GCSF) was 6 mg/m2. Ten pts have been treated with SB-921 (+GCSF) at 6 (n=4), 7 (n=3) and 8 (n=3) mg/m2, with no DLT. Enrollment at 9 mg/m2 is ongoing. Among the first 39 pts treated with SB-921 (−GCSF), mean age was 47 yr, 49% were male, histology was 46% HL, 28% aNHL, and 26% iNHL, 67% had ≥3 prior CT regimens. The most common Grade 3/4 adverse event (AE) was neutropenia (42% of pts treated at or above MTD). Other Grade 3/4 AEs were uncommon. No neuropathy or alopecia >Grade 1 was reported. Demographics and AEs in the (+GCSF) cohort are similar with less Grade 3/4 neutropenia. Two partial responses (PRs) have been reported, both in elderly pts with HL, 1 at 6 mg/m2 (−GCSF) after 2 cycles and 1 at 8 mg/m2 (+GCSF) after 2 cycles.

Conclusions: The MTD of SB-921 (−GCSF) on a d1/d15 q28d schedule was 6 mg/m2 (dose density = 0.42 mg/m2/d). The current MTD (+GCSF) is ≥8 mg/m2 and dose escalation is continuing. This dose density (0.57 mg/m2/d) is nearly 3-fold higher than observed in the FIH trial with a q21d schedule (0.2 mg/m2/d). SB-921 is well tolerated with few Grade 3/4 AEs other than hematologic. Activity has been observed in HL, with 2 PRs at doses ≥ the (−GCSF) MTD.
Tolerability and Efficacy of Bortezomib Added to CVP-R for Previously Untreated Advanced Stage Follicular Lymphoma: Interim Analysis of a Phase II Study by the NCIC Clinical Trials Group.
Authors: Sehn Laurie H , Macdonald David A , Rubin Sheldon H. , Rubinger Morel , Imrie Kevin R , Chapman Judy-Anne W , Djurfeldt Marina , Shepherd Lois , Couban Stephen , Crump Michael
Published: April 2020

Background: Despite recent improvements in therapy, follicular lymphoma (FL) remains incurable with standard treatment, warranting investigation of new approaches. Bortezomib, the first-in-class proteasome inhibitor has demonstrated promising efficacy as a single agent in heavily pretreated patients (pts) with FL. This is the first study to evaluate the safety and efficacy of the addition of bortezomib to cyclophosphamide, vincristine, prednisone and rituximab (CVP-R).

Methods: This is a phase II multi-center open-label trial adding bortezomib (1.3 mg/m2 day 1&8) to standard dose C(750 mg/m2) V(1.4 mg/m2, capped at 2 mg) P(40 mg/m2 × 5) –R(375 mg/m2) for up to 8 cycles in pts with newly diagnosed stage III/IV FL requiring therapy. Planned accrual is 90 patients. A two-stage design was employed with a planned interim analysis of the first 28 patients to ensure an acceptable level of neurotoxicity (defined as less than 5/28 patients with grade 3/4 neurotoxicity after the first 4 cycles) and meaningful response rate (more than 12/28 patients with a complete response following 8 cycles), prior to enrolling remaining patients.

Results: Median age of the first 28 patients was 55 years (range, 30–73). Fifty percent were male and 79% had stage IV disease. FLIPI score at study entry: low 14%, intermediate 43%, high 43%. Overall, the combination of bortezomib and CVP-R was extremely well tolerated. To date, no pts have developed grade 4 neurotoxicity and only 1/28 (4%) has developed grade 3 neurotoxicity within the first 4 cycles (neuropathic pain which resolved without need for treatment modification). The incidence of grade 1 and 2 neurotoxicity was 54% and 25% respectively. Only 3 pts discontinued therapy prematurely (2 pt refusal, 1 progressive disease). Ninety-four percent of planned bortezomib treatments in the first four cycles and 93% of vincristine doses were administered without dose reduction. Hematologic toxicity was mild, with no pts experiencing grade 3/4 anemia or thrombocytopenia. Only 2 episodes of febrile neutropenia occurred and no grade 3/4 infections were noted. Although it is too early to report on efficacy in this ongoing trial, response objectives for stage I have been met, and enrollment to stage 2 is underway.

Conclusions: The addition of bortezomib to standard dose CVP-R is very well tolerated, with an acceptable level of neurotoxicity, without compromising the delivery of bortezomib or vincristine. This ongoing study will provide toxicity and efficacy data to facilitate the development of a planned phase III trial.
Anti-Tumor Activity of the Aurora a Inhibitor MLN8237 in Diffuse Large B-Cell Lymphoma Preclinical Models.
Authors: Huck Jessica J , Zhang Mengkun , Hyer Marc L , Manfredi Mark G
Published: April 2020

Aurora A kinase is a serine/threonine protein kinase that is essential for normal transit of cells through mitosis. In many tumor types the Aurora A gene is amplified and/or the protein is over-expressed. The Aurora A small-molecule inhibitor MLN8237 demonstrated robust tumor growth inhibition in xenograft models of solid tumors grown subcutaneously (S.C.) in immunocompromised mice. Here we explored the antitumor activity of MLN8237 in models of diffuse large B-cell lymphoma (DLBCL) both in vitro and in vivo. In vivo three established DLBCL xenograft models (OCI-Ly7, OCI-Ly19, and WSU-DLCL2; all cells expressing luciferase) and a primary DLBCL tumor model PHTX-22-06 were tested using MLN8237 at different doses. Rituximab, an anti-CD20 monoclonal antibody that is active against CD20+ malignant B cells and is a standard of care agent was used for comparison. Using these model systems, tumor cells were injected either I.V. (to evaluate disseminated disease), or S.C. in severe combined immunodeficient mice (SCID). Animals were dosed orally for 21 days with MLN8237 (QD or BID) at various doses, or Rituximab dosed at 10mg/kg IV (once/week) and tumor growth inhibition was monitored using either bioluminescent imaging for the disseminated models or vernier calipers for the S.C. models. Tumor growth inhibition by MLN8237 was dose dependent with 20 mg/kg bid being the most efficacious dose (TGI>100% in both disseminated OCI-Ly19 and WSU models). All animals in the OCI-Ly19 disseminated model 20 mg/kg BID treatment group demonstrated regressions and remained disease free until the end of the study, day 65. In this study the Rituximab treated animals were euthanized on day 31 due to a high level of tumor burden. In the primary tumor model, PHTX-22-06, MLN8237 dosed at 20 mg/kg BID was also the most efficacious with a TGI of 95%. Moreover, tumor growth inhibition was durable as determined by prolonged tumor growth delay (>50 days). Significant efficacy was achieved in all models tested, whether grown as disseminated or subcutaneous models. A noted increase in durability of response was observed with MLN8237 treatment when compared with previous data from solid tumor models. In vitro, MLN8237 treatment increased levels of apoptosis in the OCI-Ly19 cells in comparison to the solid tumor cell line HCT-116 (colon). Greater Annexin V positive cells and greater cleaved PARP and Caspase-3 signals were detected in the MLN8237 treated OCI-Ly19 cells when compared to HCT-116 cells. The demonstration of robust and durable anti-tumor activity in preclinical models treated with MLN8237 provides the basis for its clinical evaluation as a treatment option for DLBCL. MLN8237 is currently in multiple Phase I clinical trials.
Adenanthin Upregulates c/ebp β Expression with Induction of Differentiation of the ATRA-Sensitive and -Resistant leukemic Cells.
Authors: Wu Ying-Li , Liu Chuan-Xu , Chen Guo-Qiang
Published: April 2020

Application of all-trans retinoic acid (ATRA) has significantly improved the outcome of patients with acute promyelocytic leukemia (APL), a unique subtype of acute myeloid leukemia (AML) characterized with the reciprocal translocation t(15;17) that results in the expression of leukemia-promoting promyelocytic leukemia-retinoic acid receptor-α (PML-RARα) protein. However, most patients usually relapsed in a short period and followed by generation of retinoid resistance. Therefore, it is imperative to explore agents to overcome the resistance. In this work, we found that adenanthin, a diterpenoid compound extracted from medicinal herbs, could induce monocytic differentiation of ATRA-sensitive APL-derived NB4 cells. Especially, the differentiation-inducing effect could also be seen in the agent-treated ATRA-resistant NB4-LR1, NB4-LR2 cells, as evidenced by the morphology and the increased expression of cell surface differentiation antigens CD11b and CD14. Based on this discovery, we also found that adenanthin significantly upregulated the expression of CCAAT enhancer–binding protein beta (C/EBPβ), but not the C/EBPα and C/EBPε at its transcription and protein levels, which was accompanied by the activation of c-Jun N-terminal kinases signal pathway. On the other hand, silence of C/EBPβ by specific small-interfering RNA abrogated the adenanthin induced differentiation in NB4 cells. All these data proposed the role of C/EBPβ in adenanthin induced differentiation. Furthermore, we investigated the potential combinational effects of the natural compound with other known differentiation-inducing agents including ATRA and 1, 25-dihydroxy vitamin D3. The results demonstrated that adenanthin strikingly enhanced ATRA (10nM) induced granulocyte differentiation and 1, 25-dihydroxy vitamin D3 (1nM) induced monocytic differentiation. These results suggest that adenanthin may be a potential antileukemia agent for the ATRA-sensitive and -resistant APL and deserves to be further investigated in the future.
The Para-Isomer of Nitric Oxide-Donating Acetylic Salicylic Acid (p-NO-ASA) Effectively Induces Cell Death in B-Cell Chronic Lymphocytic Leukemia (CLL) Cells at Low Micromolar Concentrations.
Authors: Razavi Regina , Paesler Julian , Gehrke Iris , Erdfelder Felix , Hertweck Magdalena , Filipovich Alexandra , Gandhirajan Rajesh Kumar , Hallek Michael , Kreuzer Karl Anton
Published: April 2020

Introduction: B-cell chronic lymphocytic leukemia CLL is characterized by an accumulation of mature, non functional B cells. We and others have shown that WNT/β-catenin (CTNNB1) signaling appears to be constitutively activated in these cells. Furthermore, it is already long known that several compounds related to the non-steroidal antiinflamnmatory drugs (NSAID) can inhibit CTNNB1 stability and/or function. However, so far clinical studies with such substances generated disappointing results which is likely to the fact that therapeutic plasma concentrations could not be reached without producing significant toxicities. Recently, nitric oxide donating derivatives of acetylic salicylic acid (NO-ASA) have been shown to be well tolerated in humans. In addition NO-ASA could disrupt complexation of CTNNB1 and TCF-4 in vitro, whereas the latter belongs to the transcription factors which posses a central function in mediating WNT signaling. The aim of our study was to evaluate whether the para- and meta-isomers of NO-ASA selectively induce apoptosis in CLL cells.

Methods: Primary CLL cells as well as healthy peripheral blood monocytes (PBMC) were treated with varying concentrations of p- and m-NO-ASA for different time periods. Cytotoxicity was assessed by microscopic cell viability testing and an ATP assay. Induction of apoptosis was investigated by immunoblotting of PARP, caspases 3 and 9. Further, CTNNB1 protein amount was measured by immunoblotting and expression of WNT effector proteins like cyclin D1 (CCND1), C-MYC and LEF-1 was evaluated with immunoblot analysis as well.

Results: The meta-isoform of NO-ASA did not have any effect on CLL cells whereas the para-isomer showed a selective cytotoxic effect. Mean lethal concentration (LC50) values were 4.83 μM and 4.64 μM in CLL cells, respectively. LC50 values for healthy controls were more than 25-fold higher. Immunoblot analysis revealed that p-NO-ASA cleaves PARP; caspase 3 and caspase 9, decreases CTNNB1 protein levels and downregulates WNT pathway target genes in a concentration dependent manner.

Conclusion: Our findings show that the para- but not the meta-isomer of NO-ASA induces caspase-mediated apoptosis by inhibition of WNT signaling in CLL cells. NO-ASA, consisting of a traditional molecule of ASA where the NO moiety is covalently bound via a spacer, has been shown to exhibit a lower gastric toxicity than traditional NSAIDs. Therefore p-NO-ASA might be a valuable compound for the treatment of CLL. More investigations of the exact mechanism of action and the specific difference between the positional isomers are indicated.
Berberine Induces Apoptosis in Acute Lymphoblastic Leukemia Cells through Downregulation of the MDM2 Oncoprotein.
Authors: Zhang Xiaoling , Gu Lubing , Shah Noopur , Zhou Muxiang
Published: April 2020

Berberine, an isoquinoline alkaloid derived from a plant used in Chinese herbal medicine, has a wide range of biological and pharmacological activities that include anti-proliferative and pro-apoptotic effects on human cancer. However, the mechanism of berberine’s anticancer action is not clearly defined. Herein, we report that berberine induces apoptosis in acute lymphoblastic leukemia (ALL) cells through downregulation of the MDM2 oncoprotein. In studying a set of ALL cell lines, we found that the cytotoxic and apoptotic effects of berberine were positively associated with the expression levels of MDM2 by those cells, regardless of their p53 status. However, most potent apoptosis induced by berberine was seen in ALL cells with wild-type (wt) p53 and MDM2 overexpression, while no pro-apoptotic effect of berberine was detected in MDM2-negative ALL cells that had a p53-null phenotype. Berberine induced apoptosis in wt-53/MDM2-overexpressing cells through downregulation of MDM2 and a concomitant stabilization of p53, with induction of its pro-apoptotic target PUMA. This berberine-induced p53 activation in the wt-p53/MDM2-overexpressing ALL differed from that induced by the chemotherapeutic drug doxorubicin, which activates p53 without prior MDM2 downregulation or even with a late upregulation of MDM2 due to a p53-mediated increase in transcription. The berberine’s downregulation of MDM2 in ALL cells occurred at the post-translational level through modulation of DAXX, which promotes MDM2 self-ubiquitination. Because wt-p53/MDM2-overexpressing ALL cells are commonly chemo-resistant due to MDM2-regulated repression of the p53 function, the potent killing of these cells by berberine through direct targeting of MDM2 suggests that this agent may be a novel therapeutic for refractory ALL.
HERG K+ Channel Is Essential for Leukemic Cell Migration Induced by SDF-1.
Authors: Li Huiyu , Du Yi-Mei , Guo Linlin , Guo Tiannan , Jie Shenghua , Du Wen , Chen Xiangjun , Liu Wei , Zhang Jiahua , Fan Linlin , Zou Anruo , Zhang Songyu , Huang Shiang
Published: April 2020

Background: Recent studies suggest that HERG K+ channel is an important regulator of non excitable cell proliferation and migration, and has been found in tumor cells including acute myeloid leukemia(AML), where HERG K+ channel is generally considered to be absent from their healthy counterparts. Bone marrow stromal cells constitutively secrete the stromal cell-derived factor-1 (SDF-1) which is a homeostatic chemokine that signals through CXCR4, SDF-1/CXCR4 axis and plays an important role in hematopoiesis development and leukemic cells migration. In this study, we investigated whether SDF-1-induced leukemic cell migration associated with HERG K+ channel.

Methods: primary CD34+/CD38− leukemic stem cells (LSCs) were isolated by cell sorting using a FACS Vantage. Transwell was used to assess the effect of E-4031, a specific HERG K+ channel inhibitor, on leukemic cell migration, the lower chamber was filled with serum-free RPMI-1640 with 100ng/ml SDF-1. Flow cytometry was used to analyze the CXCR4 expression as well as phenotypical analysis of leukemia samples. HERG K+ channels were expressed in Xenopus oocyte by microinjection and the resulting currents were measured using the standard two microelectrode voltage clamp techniques.

Results: numbers of HL-60 cells with and without E-4031 treatment migrated towards SDF-1 in the lower chamber were 1.58±0.98 ×104 and 3.47±0.81 ×104 respectively, indicating E-4031 significantly blocked the cell migration induced by SDF-1. The similar results were also observed in primary leukemic cells (n=7) and leukemic stem cells(n=3). From a holding potential of −80 mV varying potentials from −70 mV to +50 mV in 10 mV increments (2s) were applied to elicit activating currents. Each pulse was followed by a constant return pulse to −50 mV (2s) to evoke outward tail currents. 100 ng/ml SDF-1 increased HERG K+ current expressed in oocytes, for example, at +50 mV, HERG current increased about 30% (n=5). The HERG K+ current increase by SDF-1 might contribute to the mechanism of SDF-1 induced leukemic cell migration. There were no significant changes of CXCR4 expression on both HL-60 cells and primary leukemic cells regardless of untreated and treated with E-4031 for 24 hours (p>0.05), suggesting that the leukemic cell migration induced by SDF-1 were specifically associated with HERG K+ channel, not by regulating CXCR4 expression.

Conclusion: the data showed that HERG K+ channel was essential for leukemic cell migration induced by SDF-1. SDF-1 enhanced herg current suggested that SDF-1 promotes leukemic cell migration. Blocking HERG K+ channel with specific inhibitor could decrease leukemic cell and leukemic stem cell migration caused by SDF-1. Prospectively, HERG K+ channel may be a potential therapeutic target with specific inhibitors in leukemia treatment.
Comparison of Fluorescence In Situ Hybridization for EGR1 Vs CSF1R for the Detection of Del(5q) in Myelodysplasia and Acute Myeloid Leukemia.
Authors: Sun Yang , Cook James R.
Published: April 2020

The detection of del(5q) in myelodysplastic syndrome (MDS) provides useful information to guide the choice of therapy, given the efficacy of lenalidomide in cases containing this abnormality. Fluorescence in situ hybridization (FISH) analysis offers the opportunity to specifically detect chromosomal abnormalities much more rapidly than metaphase cytogenetics, which may have a turnaround time of several weeks. However, it is currently unclear which chromosomal loci are the most appropriate to examine for detection of del(5q) in routine practice. The breakpoints on chromosome 5q are heterogeneous and two commonly deleted regions (CDR) have been described. The first CDR occurs in acute myeloid leukemia (AML) and high grade MDS and encompasses a region at 5q31 including the EGR1 locus. A second CDR, occurring in at least some cases reported as 5q- syndrome, centers around 5q33 and includes the CSF1R locus. We therefore examined whether FISH studies for EGR1, CSF1R, or a combination of both probes would provide the greatest clinical utility for detection of del(5q). 51 cases of myeloid neoplasms with del(5q) by metaphase cytogenetics were analyzed, including 5q- syndrome (n=8), refractory anemia with excess blasts (RAEB, n=8), refractory cytopenia with multilineage dysplasia (RCMD, n=6), MDS unclassifiable (n=1), therapy related MDS (n=1), myelodysplastic/myeloproliferative overlap syndromes (MDS/MPD, n=6), and AML (n=21). FISH studies using EGR1/D5S23, D5S721 and CSF1R/D5S23, D5S721 probes (Abbot Molecular, Abbot Park, IL) were performed on archival bone marrows (45 coverslip aspirate smears, 4 cytogenetic culture cell pellets, and 2 formalin fixed paraffin embedded clot sections). Normal ranges were established for each probe by analysis of appropriate negative control samples. Deletion of the EGR1 locus was detected in 49/51 (96%) cases, including each case of 5q- syndrome. The CSF1R locus, which could be analyzed in 48 cases, was deleted in 44 cases (92%). In cases with concordant results, a similar percentage of abnormal nuclei was identified with each probe. Two cases (1 MDS/MPD and 1 AML) displayed deletion of the EGR1 locus but a normal pattern for CSF1R. Two cases (1 AML and 1 MDS/MPD) showed no evidence of EGR1 or CSF1R deletion despite a del(5q) identified by metaphase cytogenetics. In conclusion, FISH for EGR1 is sufficient to successfully detect del(5q) in the vast majority of cases of MDS and AML containing this abnormality, including at least most cases of 5q- syndrome. Additional FISH studies for the CSF1R locus did not increase the diagnostic yield. Further studies will be required to determine if deletions of 5q involving EGR1 but not CSF1R influence the response to lenalidomide. A small number of cases of del(5q) are detected only by metaphase cytogenetics, possibly due to a small number of abnormal cells present prior to in vitro culture.
Suppression of Cyclin D 1 (CD1) by on 01910.Na Is Associated with Decreased Survival or Trisomy 8 Myelodysplastic Bone Marrow: A Potential Targetted Therapy for Trisomy 8 MDS.
Authors: Shenoy Aarthi , Pfannes Loretta , Wilhelm Francois , Maniar Manoj , Young Neal , Sloand Elaine M
Published: April 2020

CD34 positive cells from patients with trisomy 8 myelodysplastic syndrome (MDS) have pronounced expression of early apoptotic markers compared to normal hematopoietic cells. However, trisomy 8 clones persist in patients with bone marrow failure and expand following immunosuppression (Sloand EM et al; Blood2005; 106(3):841). We have demonstrated up-regulation of c-myc, survivin, and CD1 in CD34 cells of patients with trisomy 8 (Sloand et al; Blood2007; 109(6):2399). Employing siRNA mediated knockdown of the anti-apoptotic protein survivin, we demonstrated a decrease in trisomy 8 cell growth and postulated that increased Cyclin D1 caused the upregulation of survivin resulting in resistance of these cells to apoptosis.

Using fluorescent in situ hybridization (FISH) we showed that the novel styryl sulfone, ON 01910.Na (Vedula MS et al; European Journal of Medicinal Chemistry2003;38:811), inhibits cyclin D1 accumulation and is selectively toxic to trisomy 8 cells while promoting maturation of diploid cells. Flow cytometry of cultured cells demonstrated increased proportions of mature CD15 positive myeloid cells and decreased number of immature CD33+ cells or CD34+ blasts (Sloand EM et al; Blood2007;110:822).

These encouraging in vitro data led to a phase I/II trial of ON 01910.Na in MDS patients with refractory anemia with excess blasts who had IPSS =/> int-2. This study was designed to assess the safety, and activity of escalating doses of ON 01910.Na (800 mg/m2/day × 3 days, 800 mg/m2/day × 5 days, 1500 mg/m2/day × 5 days, 1800 mg/m2/day × 5 days every 2 weeks) in MDS patients. To date five MDS patients have been treated with ON 01910.Na for 4 to 16 weeks in the first two dose cohorts. Two patients had isolated trisomy 8, two had complex cytogenetic abnormalities including trisomy 8 in all aneuploid cells, and one had monosomy 7. Three and five day infusions were well tolerated. Pharmakokinetic analysis showed that the half life of the drug is 1.3 ± 0.5 hours without signs of drug accumulation. Four of five patients demonstrated a rapid and significant decrease in the number of peripheral blasts and aneuploid cells after 4 weeks of therapy (see below), concomitantly with increases in neutrophil and/or platelet counts in four patients. All four patients exhibiting a biological effect of drug treatment had trisomy 8 in their aneuploid clone prior to therapy. One monosomy 7 patient, previously refractory to EPO became responsive to Darbopoietin and another trisomy 8 patient became platelet-transfusion independent.

In this early safety trial, ON 01910.Na demonstrates efficacy at early timepoints with respect to improved cytopenias and decreased blast counts. Continued enrollment and long term follow-up will further detail clinical efficacy and impact on the long term prognosis of high risk MDS patients treat with this drug.

Figure Figure
Characteristics and Outcome of MDS with Isolated Del 20q: The GFM Experience in 64 Cases.
Authors: Braun Thorsten , de Botton Stephane , Taksin Anne-Laure , Park Sophie , Beyne-Rauzy Odile , Chevret Sylvie , Fontenay Michaela , Cassinat Bruno , Lazareth Anne , Eclache Virginie , Terre Chrisitine , Vey Norbert , Guerci Agnes , Dreyfus Francois , Bordessoule Dominique , Stamatoullas Aspasia , Castaigne Sylvie , Fenaux Pierre , Ades Lionel
Published: April 2020

Background: Isolated del 20q is common in MDS, and considered of good prognosis in the IPSS, but no large series of MDS with del 20q have been reported.

Methods: We studied characteristics and outcome of 64 MDS patients (pts) with isolated del 20q diagnosed between 1990 and 2008 in 5 centers of the GFM. They were also compared with MDS without del 20q included in the GFM registry (containing 995 karyotyped MDS diagnosed since 2003).

Results: Median age of the 64 MDS with isolated del 20q was 72 (48–92), with 41 males and 23 females. They included 34 RA, 8 RARS, 16 RAEB, 4 CMML and 2 RAEB-T. Fifty two percent of them had less than 5% of marrow blasts, compared to 34% in the non del 20q pts included in the GFM registry (p=0.006). Despite a significantly higher proportion of low and int-1 IPSS risk (90% vs 78%, p=0.021) the isolated del 20q cohort had lower platelet count (mean 156 vs 212 G/l, p=0.014) and higher reticulocyte count (mean 84 vs 52 G/l, p=0.012) than non del 20 q pts included in the GFM. No significant difference was found for age, gender, FAB, ANC and Hb level between isolated del 20q and non del 20q pts. When the analysis was restricted to pts with less than 5% marrow blasts, the same differences were seen between isolated del 20q and non del 20q pts. On the other hand, 12 (19%) of the pts with isolated del 20q had Hb > 12g/dl and platelets < 100 G/L vs 80 of non del 20q patients (p=0.007). The 12 pts with isolated del 20q and plts <100G/L without anemia, were mostly men (9/12), with marrow blasts <5% in 83% (10/12), and all (100%) had ANC >1 G/l. None of those del 20q pts progressed to AML (compared to 17% of isolated del 20 q pts with Hb <12 g/dl and/or plts >100 G/l) and their median overall survival was 39 months vs 29 months, respectively (p=0.32). Median survival of MDS with isolated del 20q was 37 months and was significantly longer in pts with marrow blasts <5% (12 vs 60 months, p=0,0001), with low and int-1 IPSS score (39 vs 11 months, p=0.01) and with Hb >11 g/dl (53 vs 16 months, p=0.002). Survival could not be compared between isolated del 20q and non del 20q pts because of the short observation period of the GFM registry.

Conclusion: MDS with isolated del 20q are characterized by usual absence of excess of blasts lower platelets and less anemia than other MDS. About 20% of them present with isolated thrombocytopenia. Prognosis is favorable only in the absence of excess of blasts.
Large Scale Evaluation of Genetic Variation and the Risk of Multiple Myeloma.
Authors: Berndt Sonja I , Johnson David C , Crowley John , Durie Brian G , Hoover Robert , Katz Michael , Rothman Nathaniel , Van Ness Brian G. , Baris Dalsu , Morgan Gareth J
Published: April 2020

Genetic factors are thought to influence susceptibility to multiple myeloma, but most published studies to date have been small and limited in scope. To identify genetic polymorphisms associated with myeloma risk, we conducted a case-control study of 976 Caucasian myeloma cases enrolled from clinical trials as part of the International Myeloma Foundation’s Bank On A Cure® initiative and 3692 Caucasian controls from the three cohorts [Nurses’ Health Study (NHS), Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial (PLCO), and 1958 British Birth Cohort (BC58)] with genome-wide scan data. A candidate gene approach was taken with a preference given to single nucleotide polymorphisms (SNPs) in coding or regulatory regions. A total of 1097 SNPs with a minor allele frequency ≥1% were genotyped in the cases and at least one control population. In order to increase our statistical power, SNPs not genotyped in NHS and PLCO were imputed from the genome scan with MACH using the HapMap CEU population as a referent and included in the analysis if the quality control r2 was high (r2 ≥0.9). Logistic regression was used to estimate the odds ratios (ORs) and 95% confidence intervals (95% CIs) adjusting for age, sex, and country as appropriate. We found 26 loci to be associated with myeloma risk with P < 0.01. Of particular interest, we observed an increased risk of myeloma with variants in two genes involved in the metabolism of pyrimidines, DPYD and MTHFR. An increased risk of myeloma was found with two independent SNPs, rs1023244 and rs1399291, in DPYD (ORperGallele = 1.43, 95% CI: 1.16–1.76, P = 0.0008 and ORperTallele = 1.18, 95% CI: 1.06–1.31, P = 0.003, respectively) and with the MTHFR high activity 677C allele (rs1801133, ORperC allele = 1.18, 95% CI: 1.05–1.33, P = 0.006). We also observed significant associations for nonsynonymous SNPs in genes involved in cell cycle checkpoint regulation (ATR, P = 0.009; ZAK, P = 0.007) and the DNA damage bypass pathway (REV3L, P = 0.008), suggesting that alterations in DNA damage mediation may modulate myeloma susceptibility. In conclusion, this large study found SNPs in several pathways, including pyrimidine metabolism and DNA damage mediation, to be associated with myeloma risk. Additional studies are needed to replicate these findings and to further explore genetic variation in these regions.
Wnt3 Signaling Augments integrinβ1-Dependent Adhesion-Mediated Drug Resistance of Multiple Myeloma in Autocrine and Paracrine Manner.
Authors: Kobune Masayoshi , Murase Kazuyuki , Iyama Satoshi , Sato Tsutomu , Kawano Yutaka , Kikuchi Shohei , Miyanishi Koji , Takimoto Rishu , Kato Junji
Published: April 2020

Adhesion of myeloma cells to BM stromal cells is now considered to play a critical role in chemo-resistance. However, little is known about the molecular mechanism of cell adhesion mediated drug resistance (CAM-DR) in MM. In this study, we focused on relationship between drug resistance and expression of Wnts, the factor regulating the cell adhesion and proliferation, in myeloma cells. To gain insight into involvement of Wnt signaling in CAM-DR, we first screened the expression of Wnt family in myeloma cell lines (RPMI8226, ARH77, KMS-5, and MM1S) by reverse transcription-polymerase chain reaction analysis. Although the mRNAs of Wnt2b, Wnt7a and Wnt10b were variably expressed in some of myeloma cell lines, Wnt3 mRNA was detected in all the myeloma cells examined. KMS-5 and ARH77, which highly expressed Wnt3 protein, tightly adhered to human BM stromal cells and accumulation of beta-catenin and GTP-bounded RhoA was observed in these myeloma cell lines. Conversely, RPMI8226 and MM1S, which modestly expressed Wnt3 protein, rather weakly adhered to human BM stromal cells. We then examined the relevance of Wnt3 expression to adhesive property to stromal cells and to CAM-DR of myeloma cells. KMS-5 and ARH-77 exhibited apparent CAM-DR against Doxorubicin. This CAM-DR was significantly reduced by anti-integrinβ1 antibody, a Wnt-receptor competitor, secreted Frizzled related protein-1 and Rho kinase inhibitor (Y27632 and OH-fasudil), but not by the specific inhibitor of canonical signaling (DKK-1), indicating that Wnt-mediated CAM-DR which is dependent on integrinβ1- mediated attachment to stromal cells is induced by Wnt/Rho pathway signal. This CAM-DR for doxorubicin was also significantly reduced by Wnt3 siRNA transfer to KMS-5. On the other hand, the cell adhesion of MM1S was dramatically augmented by addition of Wnt3 containing conditioned medium (CM) and suppressed by integrinα4 or β1 antibody (VLA4)(Fig 1). Furthermore, CAM-DR of MM1S was significantly augmented by Wnt3 CM or adhesion of mesenchymal stem cells which expressed Wnt3, but not BM stromal cells which did not express Wnt3 mRNA. These results suggest that adhesion of myeloma cells on stromal cells was regulated by Wnt signaling in autocrine or paracrine manner. The Wnt3 signaling pathway could be a promising molecular target to overcome CAM-DR.
Glucocorticoid Receptor Expression Correlates with Clinical Outcome in Myeloma Patients Treated with Glucocorticoid-Containing Regimens.
Authors: Ma Shuo , Zhan Fenghuang , Ma Chunguang , Tessel Michael , Zhang Lijun , Rajkumar S. Vincent , Singhal Seema , Barlogie Bart , Shaughnessy John , Rosen Steven T. , Krett Nancy
Published: April 2020

Glucocorticoid (GC) is an effective anti-myeloma agent by directly inducing apoptosis and growth inhibition in myeloma cells. The anti-myeloma action of GC is mediated by its intracellular receptor, the glucocorticoid receptor (GR). The response to GC is variable among myeloma patients and resistance to glucocorticoid treatment develops almost invariably. The mechanism regulating glucocorticoid-sensitivity in not well understood, although it has been hypothesized that the regulation might occur at both the receptor (GR) and post-receptor level. In MM.1 myeloma cell lines, we observed significant down-regulation of GR expression as the cells develop GC-resistance, suggesting that GR expression level is an important determinant of GC-response. Here we present evidence that GR expression level directly correlates with the clinical outcome in myeloma patients who were treated with GC-containing regimens. First, using a quantitative real-time PCR analysis, we performed a retrospective analysis of GR gene expression on bone marrow plasma cell samples from 35 patients with newly diagnosed myeloma who were then treated on the ECOG study E1A00. This is a phase 3 trial which randomized patients to dexamethasone (dex) alone vs. dex in combination with thalidomide. Pre-treatment expression levels of GR-alpha, GR-beta and GR-P splicing isoforms were measured for 35 patients, of whom, 10 had post-treatment samples available. Interestingly, a large variation of the GR expression levels was observed across the patient samples, with the GR-alpha being the predominant isoform. However, a clear correlation between GR expression and treatment response was not seen, likely due to the small sample size. To access a larger patient pool, we retrospectively analyzed the GR expression levels using the cDNA microarray data from the Arkansas group on 351 newly diagnosed myeloma patients treated with Total Therapy 2, of which dexamethasone was an essential component throughout the treatment course. Of the 351 patients, both event-free (EFS) and overall survivals (OS) were compared between 49 patients (14%) with low levels of GR expression and the rest (86%) with high levels of GR expression. Strikingly, the cases with low levels of GR expression were associated with statistically significantly worse EFS and OS compared to those with high levels of GR expression, with the 5-year EFS of 20% vs. 52% and OS of 34% vs. 68%, P < 0.01. In summary, we have shown for the first time that the baseline level of glucocorticoid receptor expression in newly diagnosed myeloma patients predicts their clinical response to GC-containing regimen and the overall clinical outcome.
The CXCR4 Antagonist AMD3100 Stimulates the Proliferation of Myeloma Cells.
Authors: Kim Ha-Yon , Kim Seong-Woo , Lee Hyo-Jin , Yun Hwan-Jung , Kim Samyong , Jo Deog-Yeon
Published: April 2020

AMD3100, an antagonist of the chemokine receptor CXCR4, is about to be used clinically for the peripheral mobilization of hematopoietic stem cells, especially in patients with lymphoma and multiple myeloma. However, AMD3100 has been shown to activate a G protein coupled with CXCR4 and thus acts as a partial CXCR4 agonist in vitro. Although stromal cell-derived factor-1 (SDF-1) alone has minimal or negligible effects on the growth and survival of myeloma cells in vitro, many reports are consistent with the SDF-1/CXCR4 axis being involved in the progression of myeloma. Therefore, it is necessary to address the question of whether AMD3100 functions as a partial agonist for CXCR4 in myeloma cells, before it is released for wide clinical application. In this study, we explored whether AMD3100 affects the proliferation and survival of myeloma cells in vitro. As demonstrated previously, AMD3100 markedly inhibited the SDF-1 induced chemotaxis of myeloma cells, including three cell lines (RPMI8226, U266, and ARH77 cells), and CD138+ primary human myeloma cells. AMD3100 also induced internalization of CXCR4. SDF-1 alone did not stimulate the proliferation of these myeloma cells, nor did it rescue the cells from apoptosis induced by serum deprivation. By contrast, AMD3100 at 10−5M enhanced the proliferation of all three myeloma cell lines in serum-free condition by up to 2-folds in 3-day cultures, which was abrogated by pretreating the cells with pertussis toxin (PTX). This phenomenon was also observed with CD138+ primary human myeloma cells. In addition, AMD3100 enhanced the proliferation of U266 and ARH77 cells induced by interleukin-6 (IL-6). AMD3100 partially inhibited the apoptosis induced by serum deprivation, and the anti-apoptotic effect of AMD3100 was further enhanced in the presence of IL-6. AMD3100 on its own induced the phosphorylation of Akt and ERK1/2 but not Stat3 and p38/MAPK in RPMI8226 and U266 cells. The phosphorylation was also inhibited by pretreating the cells with PTX. The signal blocking agents wortmannin, PD98056, SB203580, and rapamycin did not affect the AMD3100-induced proliferation of RPMI8226 cells. LY294002 at concentrations not inhibiting spontaneous proliferation (5 μM or less) did not affect AMD3100-induced proliferation of the cells. AG490 inhibited spontaneous proliferation and AMD3100-induced proliferation of the cells in a dose-dependent manner. Accordingly, the signal-blocking effects were unclear. However, these results show that AMD3100 partially overcame the strong growth inhibition of myeloma cells induced by AG490. Our results indicate that AMD3100 can induce the phosphorylation of signaling molecules and stimulate the proliferation of myeloma cells, through signaling via G-protein pathway.
Proteomic Profiling of Multiple Myeloma: Correlation of Protein and Gene Expression Data.
Authors: Edmondson Ricky D , Thyparambil Sheeno P , MacLeod Veronica , Barlogie Bart , Shaughnessy John D.
Published: April 2020

Although melphalan-based autologous stem cell transplantation has improved prognosis for patients diagnosed with Multiple Myeloma, survival varies from a few months to more than 15 years with an individual’s risk not accurately predicted with standard prognostic variables. Correlating genome-wide mRNA expression profiles in purified myeloma cells with outcome, we recently showed that that the differential expression of 70 genes could identify patients at high risk for early disease related death [1]. The utility of a high throughput proteomics platform in the analysis of clinical samples has great potential but as of yet none have been firmly established. Herein, we describe the use of such a platform and its utility in stratifying patients with Multiple Myeloma in terms of high and low risk disease. Preliminary analysis indicates that the proteomics data can separate the patients into risk groups, although the proteins responsible for the assignment are not identical to the 70 genes identified in the gene expression profiling experiments. In addition to the proteomic analysis of plasma cells enriched using anti-CD138 immunomagnetic beads from mononuclear cell fractions of bone marrow aspirates from newly diagnosed myeloma patients; we have performed (in triplicate) LCMS profiling on plasma cells from 30 patients isolated prior to and 48 hours after a single test-dose application of bortezomib at 1.0mg/m2. An aliquot of 100,000 plasma cells was enzymatically digested with trypsin and a fraction (~5,000 cells) analyzed using our proteomics platform (an Eksigent nanoHPLC coupled to a ThermoElectron LTQ-Orbitrap with data analyzed using the Elucidator software package from Rosetta Biosoftware). The correlation of the proteomic profiles to gene expression profiles and clinical parameters will be presented. The analysis of proteins that were observed to change (p<0.01) in abundance after the single agent dose of the proteasome inhibitor bortezomib yielded an unanticipated finding; the abundance of 30 proteins associated with the proteasome were observed to increase in a subset of patients. The majority of the patients with the increased levels of proteasome related proteins are predicted by GEP to have high risk disease. The proteomic data will be discussed in terms of its utility in the identification of activated pathways as well as in the development of a prognostic indicator as was achieved using gene expression profiling.
Agonist Anti-TRAILR1 Human Mab, Hgs-ETR1, Induces Myeloma Cell Death Mainly through the Intrinsic Pathway of Apoptosis.
Authors: Ménoret Emmanuelle , Surget Sylvanie , Gomez-Bougie Patricia , Bataille Régis , Amiot Martine , Pellat-Deceunynck Catherine
Published: April 2020

We previously reported that agonist anti-TRAILR mAbs (HGS-ETR1) induced cell death of myeloma cells through both the extrinsic and intrinsic pathway. Although caspase 8 activation was fully required to initiate death signaling, it was unclear whether intrinsic pathway played or not a major role in apoptosis execution. In the current work, we evaluated the contribution of the intrinsic pathway in a large panel of HMCL (n=10). To address this question, we monitored mitochondrial depolarization and the contribution of caspase 9. Apoptosis and mitochondrial polarization were both evaluated by flow cytometry. Kinetic analysis of mitochondrial polarization (JC-1) shows that all mitochondria were fully depolarized after only 4h following HGS-ETR1 mAb addition: the depolarization started early, as soon as 1h. In good agreement with mitochondrial depolarization, we observed that specific inhibitor of caspase 9 activity (z-LEHD-fmk) always prevented at least 50% of death (and up to 90%). Our data show that in all cell lines evaluated, activation of the intrinsic pathway was required for cell death even with high concentrations of mAb. We previously showed that anti-TRAILR apoptosis was always associated with an early and massive Mcl-1 cleavage (Menoret et al Blood2006, 108:1346). In myeloma cells, Mcl-1 is physically associated with Bim and thus prevents Bim to induce mitochondrial cell death (Gomez-Bougie EJI 2004, 34;3156). By immunoprecipitation, we show that, in the presence of HGS-ETR1 mAb, Mcl-1/Bim complexes were disrupted and Bim was free to activate Bax. Bim is also found to be complexed to both Bcl-XL and Bcl-2 in viable cells. During anti-TRAILR mAb induced apoptosis, Bcl-2 and Bcl-XL were also cleaved and full length Bcl-XL, as Mcl-1, fully disappeared indicating that BcL-XL decrease probably participated to the increase of free Bim. Furthermore, immunoprecipitation of Bax showed that Bax was associated with Bcl-2 (and not with Mcl-1 or Bcl-XL) and this association was reduced in apoptotic conditions. These data show that apoptosis induced by anti-TRAILR mAb mainly involved the intrinsic pathway through disruption of both anti-apoptotic/Bim and Bcl-2/bax complexes. This result suggests that the association of anti-TRAILR mAb with melphalan or bortezomib would not synergistically increase anti-TRAILR mAb efficiency and that anti-TRAILR mAb should be rather combined with drugs acting independently of the intrinsic pathway.
A Humanized Anti-Ganglioside GM2 Antibody, BIW-8962, Exhibits ADCC/CDC Activity against Multiple Myeloma Cells and Potent Anti- Tumor Activity in Mouse Xenograft Models.
Authors: Ishii Toshihiko , Chanan-Khan Asher Alban , Jafferjee Jazur , Ersing Noreen , Takahashi Takeshi , Mizutani Masato , Shiotsu Yukimasa , Hanai Nobuo
Published: April 2020

BIW-8962 is a humanized anti-ganglioside GM2 (GM2) monoclonal antibody, produced by Poteligent technology to enhance ADCC activity. GM2 is expressed on many cancer cells including multiple myeloma (MM), small cell lung cancer and glioma cells. In this study, we evaluated the anti-myeloma activity of BIW-8962 in preclinical myeloma models both in vitro and in vivo. Expression of GM2 was analyzed in 15 human MM cell lines by FCM. Eleven out of 15 MM cell lines had positive surface expression of GM2. GM2 as a potential target was then verified in primary MM samples obtained from patients. Eleven out of 15 samples were positive for GM2. We then used two GM2 positive MM cell lines (U266B1 and KMS-11) and evaluated ADCC and CDC activity of BIW-8962 in vitro. BIW-8962 exhibited a potent ADCC and less potent CDC activity. In vivo anti-tumor activity of BIW-8962 was then examined using the standard subcutaneous xenograft model; KMS-11 was inoculated in the flank of SCID mice. BIW-8962 (intravenously administered biweekly for 3 weeks) exhibited a potent anti-tumor activity from as low a dose level as 0.1 mg/kg. Furthermore, in a more clinically relevant model, in which OPM-2/GFP (GM2 positive MM cell line) cells were intravenously inoculated into SCID mice with preferentially tumor growth within the bone marrow microenvironment, BIW-8962 (intravenously administered biweekly for 4 weeks, 10 mg/kg) suppressed OPM-2/GFP cell growth and serum M protein elevation, demonstrating in vivo anti-myeloma effect of BIW-8962. Our preclinical investigations rationalize clinical evaluation of BIW-8962 in patients with MM. Currently BIW-8962 is being investigated in a Phase 1 study in patients with multiple myeloma.