bluuebunny/crossref_metadata_2025 huggingface dataset:
Prospective Randomized Comparison of High Dose AraC and Autologous Peripheral Blood Stem Cell Transplantation as Late Consolidation for Patients ≤60 Years with Standard Risk AML.
Authors: Ganser Arnold , Krauter Jürgen , Hoelzer Dieter , Ottmann Oliver , Martin Hans , Lübbert Michael , Finke Jürgen , Heit Wolf , Fiedler Walter , Kanz Lothar , Schlimok Günter , Kirchner Hartmut , Raghavachar Aruna , Brossart Peter , Brugger Wolfram , Heil Gerhard
Published: April 2020

484 patients aged ≤60 years with de novo (n=385) or secondary (n=99) acute myeloid leukemia (AML) were treated with risk-adapted therapy. Patients with t(8;21), inv(16) or normal karyotype and good response (GR) to induction I (≤5% blasts in day 15 BM) were considered standard risk (SR), all others as high risk (HR). Induction I consisted of standard dose araC, idarubicine and etoposide (IVA-I). Patients with GR to IVA-I continued with IVA-II on day 21 as double induction. Patients with bad response (>5% blasts in day 15 BM) received FlAG/Ida as second cycle. Double induction was followed by early consolidation with intermediate dose araC. As late consolidation, SR patients with normal karyotype and an HLA-matched sibling received matched related donor (MRD) transplantation. The remaining SR patients were randomized between high dose araC (HD-araC; 12 x 3g/m2)/daunorubicine or an autologous peripheral blood stem cell transplantation (autoPBCSCT) with PBSC mobilized after early consolidation. 90% of SR patients (n=250) achieved complete remission (CR) in contrast to 58% of HR patients (n=234) (overall CR rate 75%). 4% of the SR patients died during induction. At 70 months, overall survival (OS) was significantly better for SR (51%) than for HR patients (28%). Within the SR group, relapse free survival at 50 months was not different between patients with normal karyotype (n=165), inv(16) (n=30) or t(8;21) (n=30). 55 SR patients were randomized to receive HD-araC and 59 to autoPBSCT. Distribution of the main characteristics were well balanced (age, performance status, cytogenetics, FLT3 mutation status, de novo or secondary AML). At 63 months, OS was 62% for patients treated with autoPBSCT and 59% for those receiving HD-araC (p=0.91, intent-to-treat). Median duration of neutropenia (<500/μl) was 8 days for autoPBSCT and 20 days for HD-araC (p<0.05). This corresponded to a significantly higher rate of septicemia (20% vs. 10%) and pneumonia (13% vs. 3%) after HD-araC. The duration of thrombocytopenia was 22 days after HD-araC and 11 days after autoPBSCT (p<0.01). 14 patients died in the HD-araC group (4 in CR and 10 from relapse) and 14 patients died after autoPBSCT (1 in CR and 13 from relapse). In patients with normal karyotype, OS at 67 months for the SR patients receiving MRD transplantation (n = 37) was 61% with no significant difference to HD-araC or autoPBSCT. In conclusion, OS is similar in SR AML patients after autoPBSCT, HD-araC or MRD transplantation. In SR patients without an HLA-identical sibling donor, autoPBSCT instead of HD-araC is recommended for late consolidation due to the reduced treatment related toxicity.
Increased Expression of Insulin-Like Growth Factor I Is Associated with Ara-C Resistance in Leukemia.
Authors: Abe Shori , Harigae Hideo , Funato Tadao , Kameoka Junichi , Takahashi Shinichiro , Kaku Mitsuo , Sasaki Takeshi
Published: April 2020

Cytosine arabinoside (Ara-C) is one of the most common drugs used in the treatment of acute myeloid leukemia (AML). Therefore, the resistance to Ara-C is a major obstacle to improvement of the cure rate of AML. However, the molecular mechanism for Ara-C resistance has not been fully understood. In order to investigate the mechanism involved in Ara-C resistance, the gene expression profile of Ara-C-resistant human K562 leukemia cells (K562/AC cells) was compared with that of the Ara-C-sensitive K562 parental cells by using a cDNA microarray platform. Correspondence analysis demonstrated that the insulin-like growth factor I (IGF-I) gene was significantly upregulated in K562/AC cells. The increased expression of IGF-I in K562/AC cells was confirmed by quantitative RT-PCR (RQ-PCR). We then studied the biological significance of IGF-I upregulation in Ara-C resistance. First, K562/AC cells were treated by suramin, a nonspecific growth factor antagonist. The treatment with suramine reduced viability and induced apoptosis of K562/AC cells. Furthermore, the addition of IGF-I neutralizing antibody exhibited the same effect as suramine. Moreover, the IC50 of Ara-C sensitive K562 parental cells was increased by the addition of exogenous IGF-I ligand. These results strongly suggest that IGF-I-IGF-I-R pathway is directly involved in Ara-C resistance. Next, the activation status of phosphatidylinositol 3-kinase (PI3-kinase)/Akt pathway, which is the downstream of IGF-I-IGFI-R signal, in K562/AC cells was examined. Western blot analysis showed that the phosphorylation level of Akt is higher in K562/AC cells than K562 sensitive cells. In addition, LY294002, the specific inhibitor for PI3-kinase reduced the survival of K562/AC cells. Since activation of PI3-kinase/Akt pathway has been shown to exhibit an anti-apoptotic effect in tumor cells, it is possible that IGF-I induces Ara-C resistance by escaping from apoptosis through PI3-kinase/Akt pathway in an autocrine or paracrine fashion. Finally, the expression level of IGF-I was examined in 27 clinical samples by RQ-PCR. The results showed that IGF-I expression level was higher in leukemic cells in refractory or relapsed AML patients, who received Ara-C combined chemotherapy, than that at diagnosis. These results suggest that the inhibition of the IGF-I-IGF-I receptor pathway is a valuable therapeutic approach to overcome Ara-C resistance in AML.
Reversal of Rituximab-Resistant AIDS-B-NHL Clone to Chemotherapeutic Drug-Induced Apoptosis by Bortezomib and DHMEQ.
Authors: Vega Mario , Jazirehi Ali , Bonavida Benjamin
Published: April 2020

The mechanisms underlying the failure of B-NHL cancer patients to respond to treatment with rituximab, alone or in combination with chemotherapy, are not known. In efforts to address this issue, we have generated rituximab-resistant clones of the AIDS NHL cell line, (2F7RR). Recent findings have demonstrated that treatment of the wild type (wt) 2F7 with rituximab sensitized the tumor cells to various chemotherapeutic drug-induced apoptosis. Chemosensitization was the result of rituximab-mediated inhibition of the p38 MAPK signaling pathway and the selective inhibition of the anti-apoptotic Bcl-2 gene product (Vega et. al., Oncogene23:4993, 2004). Analysis of one clone, 2F7RR1, revealed that the cells have diminished surface CD20 expression and failed to respond to CDC and to apoptosis following cross-linking. In addition, the cells were resistant to rituximab-mediated chemosensitization. In contrast to wt2F7, molecular analysis of the 2F7RR1 clone revealed that rituximab failed to inhibit p-Lyn, p38-MAPK, BclXL, and Bcl-2. In addition, rituximab failed to inhibit the transcription factors NF-κB, YY1, SP-1, and STAT3. Noteworthy, 2F7RR1 exhibited higher resistance to drug-induced apoptosis compared to wt2F7 and showed overexpression of Bcl-2. Previous findings with the wt2F7 demonstrated that Bcl-2 was responsible for chemoresistance. Accordingly, we examined whether inhibition of Bcl-2 in 2F7RR1 can reverse chemoresistance. Since Bcl-2 is under the transcriptional regulation of NF-κB, we examined the effect of the NF-κB inhibitors Bortezomib and DHMEQ (DHMEQ was a kind gift from Dr.K. Umezawa Keio University, Japan) (Kikuchi, et al., Cancer Review, 2003, 63:107). The findings revealed that treatment of 2F7RR1 with these inhibitors resulted in the reversal of resistance to a number of chemotherapeutic drugs (examples: taxol, vincristine, ADR, CDDP, VP16, etc.). The chemo-sensitization by Bortezomib and DHMEQ was equivalent and the combination treatment of each of these inhibitors with the drug was synergistic. These studies present evidence that rituximab and drug-resistant tumor cells may be sensitized to chemotherapeutic drug-induced apoptosis via inhibition of NF-κB or Bcl-2. These findings also suggest that Bortezomib and DHMEQ may be clinically relevant in the treatment of rituximab and drug-resistant AIDS-B-NHL.
Microarray Analysis of Differentially Expressed Genes in Response to Retrovirally Mediated Overexpression of MDR1.
Authors: Maier Patrick , Herskind Carsten , Li Li , Laufs Stephanie , Zeller Jens W. , Fleckenstein Katharina , Wenz Frederik , Fruehauf Stefan
Published: April 2020

An important strategy for improving cancer therapy is the protection of healthy cells against therapy-induced damage. In this context gene therapy can complement radio- and chemotherapy in the treatment of tumours. Retroviral overexpression of P-glycoprotein (P-gp), the product of the MDR1 (multidrug resistance 1) gene, can deliver such a protective effect. P-gp possesses two relevant functions: its detoxifying effect acts as an efflux pump and could be important in high-dose chemotherapy, the anti-apoptotic effect of overexpressed P-gp could protect normal cells in tumour radio- or chemotherapy. The protection of haematopoietic stem cells in lymphoma, myeloma or solid tumor therapy is a promising candidate for this approach. However, little is known which other gene expressions might be influenced by MDR1-overexpression. Therefore we analyzed differentially expressed genes in the human lymphoblastoid cell line TK6 transduced with onco-retrovirus SF91m3 encoding MDR1 using the GeneChip Human Genome U133 Plus2.0 (Affymetrix). 61 annotated genes showed a significant change in expression (p<10−4) in MDR1 overexpressing compared to control transduced (overexpression the neomycin resistance gene) and untransduced cells. We found that genes coding for detoxifying and exocytotis proteins (e.g. ABCB4, ALDH1A, unc13) were up-regulated. Furthermore several proapoptotic genes were down-regulated (e.g. Casp1, Casp4) with concomitant increased expression of antiapoptotic genes (e.g. Akt3). The differential expressions for several genes were verified with real-time PCR. The results confirmed the dependence of these gene regulations on MDR1 overexpression. The influence of MDR1/P-gp overexpression on apoptotic signalling and cell survival was further corroborated by reduced apoptosis rates in response to irradiation in SF91m3 transduced cells compared to the control cell lines. Our genomics results support the findings from other studies that overexpression of MDR1 confers protection against apoptotic stimuli. The multidrug resistance phenotype does not seem to be only due to high P-gp expression but also to other metabolic proteins. Our results could have important implications for MDR1-gene therapy in patients receiving chemotherapy regimens in combination with radiotherapy.
High Incidence of Myelodysplasia and Secondary Leukemia in the UK MRC Pilot Study of Autografting in Chronic Lymphocytic Leukemia.
Authors: Milligan Donald , Kochethu Geothy , Dearden Claire , Matutes Estella , MacConkey Christopher , Catovsky Daniel
Published: April 2020

In the MRC CLL Pilot Study of autografting conducted between 1996 and 2001 115 newly diagnosed patients were treated with a median of 6 cycles of fludarabine (range 3–8). In addition 26 patients received further treatment, mainly the addition of cyclophosphamide to fludarabine, CHOP or alemtuzumab, in order to achieve either CR, nPR or PR. Progenitor cells were mobilised with cyclophosphamide 2g/m2 and G-CSF. Sixty-five patients were autografted with cyclophosphamide and TBI conditioning or BEAM (20%). The median CD34 cell dose for transplanted patients was 2.6x106/Kg (range 0.75–3.9). Ten patients have subsequently developed AML/MDS at a median 3.5 years after starting treatment (range 2.5–7 years). Eight patients had undergone an autograft and 2 had received fludarabine alone and not proceeded to transplant. The actuarial risk of developing MDS/AML post autograft was 12.4% at 5 years (CI 0.7–24%). At the time of diagnosis of MDS/AML all the patients had either complex cytogenetics or/and deletion of chromosomes 5 or 7. These abnormalities were not detectable by FISH on the pre-transplant harvested material. Analysis of potential risk factors (age, stage of disease, previous treatment, cell dose, conditioning type) demonstrated only age as a possible factor (p<0.05). This frequency of post transplant MDS has been reported in low grade lymphomas but not previously in autografts for CLL. We hypothesise that this patient population may be particularly at risk of secondary MDS/AML and that potential causative factors are fludarabine, a low cell dose and transplant conditioning.

Probability of developing MDS/AML after Tx Probability of developing MDS/AML after Tx
A Gene Polymorphism within the Kinase Domain of BCR-ABL and Its Effects on Sensitivity to Tyrosine Kinase Inhibitors.
Authors: Crossman Lucy C. , O’Hare Thomas , Lange Thoralf , Willis Stephanie G. , Stoffregen Eric P. , Corbin Amie S. , O’Brien Stephen G. , Heinrich Michael C. , Druker Brian J. , Middleton Peter G. , Deininger Michael W.N.
Published: April 2020

Background: Point mutations that impair drug binding are the most important mechanism of acquired resistance to imatinib. Mutations within the ATP binding loop (P-loop) of BCR-ABL are associated with a poor prognosis in patients on imatinib. We have identified a single nucleotide polymorphism at position 247 (numbering according to ABL-B) that leads to the replacement of lysine by arginine. In CML patients, either the lysine or arginine allele of amino acid 247 becomes part of the BCR-ABL fusion gene. Due to its close proximity to the P-loop, we decided to investigate the allele frequency of K247R and whether the presence of the arginine allele affected sensitivity of Bcr-Abl to Abl kinase inhibitors.

Patients and Methods: We investigated the frequency of the arginine allele of K247R in 157 patients with CML and 213 healthy blood donors by conventional sequencing, restriction enzyme digestion and single strand conformational polymorphism analysis. We used Abl autophosphorylation and substrate phosphorylation assays, immunoblotting and cellular proliferation assays to examine the influence of K247R upon imatinib and dasatinib sensitivity.

Results: We found that frequency of the arginine allele of K247R was 1.6% in patients with CML and 0.2% in the controls (P = 0.11). In one patient analysis of CD3+ and CD33+ obtained at the time of complete cytogenetic response revealed the presence of K247R in both cell compartments, consistent with a polymorphism. Three out of 5 patients with the arginine allele of K247R expressed in BCR-ABL failed to achieve a major cytogenetic response to imatinib, suggesting reduced drug sensitivity. However, in vitro assays showed no alteration in sensitivity to imatinib or dasatinib compared to “wild type”.

Conclusions: K247R is a rare polymorphism within the kinase domain of Abl. There is no evidence that K247R is more frequent in CML patients, and the presence of K247R does not affect drug sensitivity. It is important that clinicians are aware that K247R does not reflect a kinase domain mutation, and that its presence does not signal a need to change therapeutic strategy unless there are other signs of inadequate response to drug therapy.
Characterization of Gene Expression Associated with Cyclophosphamide Resistance in Chronic Myeloid Leukemia (CML).
Authors: Bao Fei , Nordberg Mary L. , Polk Paula , Sun Amanda , Murray David , Andersson Borje S. , Munker Reinhold
Published: April 2020

Cyclophosphamide (CP) is one of the alkylating agents collectively referred to as oxazaphosphorines that are used to treat many types of cancers including myeloid leukemia. Tumor cell drug resistance is an important factor for clinical treatment failure. The mechanisms of drug resistance are multifactorial and incompletely understood. KBM-7 human CML cell line was established from blast cells from a patient in the terminal phase of CML. In the CP resistance model, the B5-180 sub-line was isolated following exposure to the in vitro active CP analog 4HC. B5-180 cells were cross-resistant to busulfan and γ-radiation. Total RNA was extracted and hybridized to Affymetrix Genechip HG-U95Av2 arrays. Each array contains 12,386 probes corresponding to approximately 9000 known human genes. Each cell line was arrayed in triplicate. Quantitative RT-PCR, Fluorescence In-Situ Hybridization (FISH) and cytogenetic analysis were performed in both cell lines. Both the KBM-7/B5 parental line and B5-180 resistant sub-line expressed high-levels of BCR-ABL transcripts by real-time RT-PCR. FISH and cytogenetic analysis revealed multiple copies of t(9;22) translocation and other additional chromosomal abnormalities such as trisomy 8, and abnormalities of chromosome 18 in both cell lines. Gene array identified 794 gene transcripts that were more than twofold (range from 2-fold to 2675-fold) over-expressed or under-expressed in the resistant line relative to the parental line. ALDH1A1 (aldehyde dehydrogenase 1 family) showed the most differential expression between sensitive and resistant cell lines, ALDH1A1 was upregulated more than 2000-fold in the resistant sub-line. ALDH-2 (aldehyde dehydrogenase 2 family mitochondrial) was also expressed substantially higher in the resistant line. This finding is consistent with the established fact that elevated ALDH activity is an important factor in the resistance of B5-180 cells to 4HC. The remaining differentially expressed genes encode proteins with a wide variety of biochemical functions, which include 44 apoptosis and 7 anti-apoptosis-related genes, 56 genes related to cell cycle and cell growth, 6 DNA repair genes, 13 genes involved in hemopoiesis and B-cell activation. We also tested the expression of the hematopietic transcription factor PU-1 and PUB, a novel PU-1 binding factor. Interestingly, the expression of PU-1 was decreased and PUB increased in the resistant clone. In conclusion, we have identified a large number of differentially expressed genes in a CP resistant cell line derived from CML blast crisis by microarray technology. Our results suggest that CP resistance is a complex phenotype that involves multiple genes and a variety of mechanisms. Real-time RT-PCR analysis and further characterization of selected genes associated with CP resistance as well as the response in vitro to tyrosine kinase inhibitors are currently under investigation.
Identification of Three Novel Chromosomal Translocation Partners Involving the Immunoglobulin Loci in Newly Diagnosed Myeloma and Human Myeloma Cell Lines.
Authors: Hanamura Ichiro , Iida Shinsuke , Ueda Ryuzo , Kuehl Michael , Cullraro Contance , Bergsagel Leif , Sawyer Jeffrey , Barlogie Bart , Shaughnessy John
Published: April 2020

Chromosomal translocations involving the immunoglobulin heavy-chain (IgH) locus at 14q32 are genetic hallmarks of human multiple myeloma (MM) and are likely primary oncogenic events in myeloma transformation. Common recurrent translocation partner genes of IgH account for 40–50% of cases and these rearrangements result in the transcriptional dysregulation of various proto-oncogenes such as CCND1, CCND3, FGFR3/MMSET, c-MAF, MAFB, MUM 1(IRF-4), c-MYC and it is becoming clear that these dysregulated genes play important roles in the pathogenesis and the treatment outcome of MM. Although the frequency of other partner genes of IgH translocations may be rare (<1%), it is possible that many genes not currently identified as translocation partners are functionally converged into several signal pathways and play important roles as well. Through global gene expression profiling (GEP) studies of a large population of newly diagnosed MM, we have recently reported that dysregulation of any one of the three D-type cyclins is seen in almost all newly diagnosed MM and most patients with high levels of CCND2 expression was highly correlated with expression of c-MAF, MAFB or FGFR3/MMSET. In order to identify novel translocation partner genes in MM we combined GEP and fluorescence in situ hybridization (FISH) analysis in a large population of newly diagnsoed myeloma. We investigated whether IGH-CCND2 and IGH-MAFA translocations are seen in patients exhibiting high levels of CCND2 expression but not expressing c-MAF, MAFB, or FGFR3/MMSET. We also looked for a breakpoint and targeted gene at a non-random breakpoint at the 11q23 locus in 20 MM patients with 11q23 abnormalities detected by conventional G-banding. Here we report evidence of an IGH-CCND2 fusion in a newly diagnosed case of MM and an IGH-MAFA fusion in a newly diagnosed MM and an MGUS and also mapped the 11q23 breakpoint to within 100 kb in a newly diagnosed MM and a human myeloma cell line, NCU-MM-1 and identified an IGL Pou2AF1 fusion in the NCU-MM-1 line. Importantly these translcoatios do not involve the MLL or ATM genes. These three genes were all highly expressed as detected by GEP or quantitive RT-PCR in the samples in which these genes were translocated to IG loci. Although the frequency of these 3 translocations are very rare, they further the concept of a critical role of CCND2 expression in myelomegenesis and identifed the transcriptional coactivator Pou2AF1, which specifically associates with either OCT1 or OCT2, in the B-cell response to antigens and required for the formation of germinal centers, as a potential new translocation target in myeloma. Finally, our strategy of combining GEP signatures and cytogenetic analyses appears to be a practical means of identifying new IG- mediated translocations in myeloma.
Frequent Gains of Chromosome Arm 1q21 in Plasma Cell Dyscrasia Detected by Fluorescence In Situ Hybridization: Incidence Increases from MGUS to Relapsed Myeloma and Is Related to Disease Progression and Prognosis.
Authors: Hanamura Ichiro , Stewart Peter , Cavallo Frederica , Burington Bart , Rasmussen Erik , Crowley John , Zhan Fenghuang , Huang Yongsheng , Barlogie Bart , Shaughnessy John
Published: April 2020

Background: High-dose therapy with autologous stem cell transplantation (HDT-ASCT), effecting complete remissions in > 40% with median overall survival exceeding 6 years, has become the standard of care for patients with multiple myeloma (MM). Poor outcome following HDT-ASCT has been linked most strongly to metaphase cytogenetic abnormalities (CA). Tandem duplications and jumping translocations of the chromosome 1q21 region is a recurrent event in advanced MM and 1q21 amplification (amp1q21) and high expression of the CKS1B gene (mapping to 1q21), both signify poor survival in MM treated with HDT-ASCT. Smoldering multiple myeloma (SMM) and monoclonal gammopathy of undetermined significance (MGUS) have many of the genetic hallmarks of overt MM, but especially MGUS rarely proceeds to MM requiring treatment. The aim of this study was determine if amp1q21 relates to disease progression and prognosis.

Patients and Methods: 467 untreated patients with MGUS (n = 14), SMM (n = 32) and MM (n = 421) as well as 42 with relapsed MM were examined by means of triple color interphase fluorescence in situ hybridization (TRI-FISH) of the CKS1B locus.

Results: The frequency of ampq21 was 0% in 14 patients with MGUS, 43% in 32 with SMM, 43% in 421 with newly diagnosed MM and 78% in 42 with relapsed MM (p<0.001; untreated vs relapsed MM). Among SMM patients, 11 of 14 (78%) with amp1q21 but only 2 of 18 (11%) without amp1q21 progressed to active MM (p<0.001) (median follow-up, 44 mo; range, 0–120 mo). In 421 MM patients treated on Total Therapy 2 (high-dose melphalan-based tandem autotransplants +/− thalidomide), 5-yr overall survival/event-free survival were 75%/61% in without amp1q21 compared to 48%/34% with amp1q21 (both p<0.001). Among 42 relapses, amp1q21 was present in 20/21 occurring on thalidomide vs 13/21 on no thalidomide (p=0.020); the median post-relapse survival in those with amp1q21 was only 11 mo vs 25 mo in patients without amp1q21 (p=0.02).

Conclusion: (1) Amp1q21 was absent in MGUS and present in >40% of symptomatic MM and SMM. (2) in SMM, amp1q21 was associated with a higher risk of conversion to MM. (3) In overt MM, amp1q21 conferred inferior event-free and overall survival.
Gains on Chromosome Arm 9q Represent a Novel and Independent Marker of Adverse Prognosis in Multiple Myeloma Patients Receiving Upfront High-Dose Chemotherapy and Autologous Stem Cell Transplantation.
Authors: Liebisch Peter , Benner Axel , Tschajka Kathrin , Wendl Christiane , Goldschmidt Hartmut , Döhner Hartmut
Published: April 2020

Introduction: Genomic aberrations represent important prognostic markers in many hematological cancers. In multiple myeloma (MM), chromosome 13q and 17p deletions (13q−, 17p−) have emerged as important outcome predictors that indicate a dismal prognosis. Other chromosomal abnormalities have been discussed as prognostic markers in this disease but came not out as independent variables when they were tested in a multivariable fashion. However, the complexity of genomic rearrangements and the clinical heterogeneity seen in malignant plasma cell disorders argue against 13q− and 17p− as the sole genomic change of prognostic relevance. The significance of chromosome arm 1q, 9q, and 11q extra copies - three frequent genomic imbalances in MM - is undetermined.

Material and Methods: 90 patients (pts.) treated with one or two cycles of high-dose chemotherapy (HD-CTX) followed by autologous stem cell transplantation (ASCT) at a single center were analyzed by tri-color FISH and five DNA probes mapping to chromosome bands 1q21, 9q34, 11q25, 13q14, and 17p13. A multivariable analysis (Cox proportional hazards regression model) including genetic and clinical variables was performed.

Results: The most frequent aberrations in the present series were (in order of decreasing prevalence): +1q (n=39/78, 50.0%), +9q (n=38/78, 48.7%), 13q− (n=42/90, 46.6%), and +11q (n=39/85, 45.9%). 17p− was identified in only 3 out of 88 patients (3.4%), while +17p were present in 13 out of 88 patients (14.8%). The median follow-up time was 37 months (m) and the median event free survival (EFS) and overall survival (OS) time from first ASCT of the entire cohort was 26 m and 71 m, respectively. Multivariable analysis including genetic aberrations, ß2-microblobulin, albumin, LDH, Salmon/Durie stage, and age at time of diagnosis revealed +9q and 13q− as the only independent predictors of EFS (p=0.003 and p=0.01, respectively). The mEFS in patients with 13q− was 19.0 m and 20.7 m in patients with +9q. In patients with concurrent +9q and 13q−, mEFS was only 12.2 m. In patients lacking these two abnormalities, mEFS was not reached. OS was not significantly influenced by any genetic or clinical variable in our series, most likely due to effective salvage treatment after relapse.

Conclusions: +9q represents a novel and independent marker of adverse prognosis in MM. A single FISH experiment applying two DNA probes allows easy and rapid assessment of outcome in patients with malignant plasma cell disorders.
Studies of the Bcl Inhibitor GX15-070 in Multiple Myeloma Demonstrate Substantial Pre-Clinical Activity.
Authors: Stewart A. Keith , Li Zhi Hua , Wen Xiao Yan , Rauw Jennifer , Pathangey Latha , Bergsagel P. Leif , Trudel Suzanne
Published: April 2020

GX15-070 is a small-molecule antagonist of the BH3-binding groove of the Bcl-2 family of proteins and can thus induce apoptosis by inhibiting the interaction between pro- and anti-apoptotic proteins. In addition, GX15-070 activates a cellular pathway, which leads to the activation of the pro-apoptotic BH3-only protein Bim, a natural cellular inhibitor of anti-apoptotic members. Together, the direct inhibition of anti-apoptotic Bcl-2 family proteins and the activation of pro-apoptotic Bim by GX15-070 can sensitize tumor cells to apoptosis. Bcl-2 family members, particularly Bcl-XL and Mcl-1 have been implicated in the survival of myeloma cells and may be responsible, in part, for drug resistance. Consequently, GX15-070 was tested against the MMRC 12 validation cell line panel, a panel of 12 standardized and annotated human myeloma cell lines (HMCLs). Western blots preformed on 8 HMCLs confirmed expression of Bcl-2, Mcl-1 and/or Bcl-XL. Basal levels of Bcl-2 and MCl-1 were relatively similar across all HMCLs studied whereas levels of Bcl-XL were variable GX15-070 inhibited the viability of all 12 myeloma cell lines as determined by MTT assay. The mean IC50 was 215 nM; range (6.4-678nM) and sensitivity did not correlate with level of Bcl-2, MCL-1 or Bcl-XL expression. On the other hand cytotoxicity to normal human blood lymphocytes, stroma and bone marrow CFUs was not observed at concentrations which were effectively cytotoxic to HMCL. Time course experiments demonstrate that apoptosis and cleavage of caspase 3 began by 12 hours and continued to increase over a 96 hour period. Co-culture with human bone marrow stroma cells (BMSCs) failed to protect HMCL from GX15-070 induced cytotoxicity. Similarly, potent MM growth factors, Interleukin 6 (IL-6) and Insulin like growth factor (IGF)-1 did not confer resistance to GX15-070. Of interest, overexpression of Bcl-2 has been shown to attenuate PS-341-induced cell death (Mitisades); however, the combination of PS-341 and GX15-070 did not reveal synergistic interaction in preliminary results. Studies looking at other drug combinations are planned. Finally, GX15-070 produced cytotoxic responses in the first primary myeloma sample tested with further testing ongoing. Mouse xenograft studies are also ongoing and will be presented. These studies indicate extensive pre-clinical activity of GX15-070 in multiple myeloma and set the stage for planned Phase II clinical trials.
BCL6 Is Regulated by p53 through a Response Element Frequently Disrupted in B-Cell Non-Hodgkin’s Lymphoma.
Authors: Margalit Ofer , Amram Hila , Amariglio Ninette , Simon Amos J. , Granot Galit , Minsky Neri , Shaklai Sigal , Shimoni Avichai , Harmelin Alon , Givol David , Shohat Mordechai , Oren Moshe , Rechavi Gideon
Published: April 2020

The BCL6 transcriptional repressor mediates survival, proliferation and differentiation blockade of B-cells during the germinal-center reaction, and is frequently misregulated in B-cell non-Hodgkin’s lymphoma (BNHL). The p53 tumor-suppressor gene plays a crucial role in tumorigenesis. In a previous study done in our laboratory, DNA microarrays were used to identify primary p53 target genes, one of which was BCL6 (Kannan et al. 2001. Oncogene20: 2225). The BCL6 intron 1 contains a region in which 3 types of genetic alterations are frequent in BNHL: chromosomal translocations, point mutations and internal deletions. We therefore defined it as TMDR - Translocations, Mutations and Deletions Region. The BCL6 gene contains a p53 response element (p53RE) residing within the TMDR. This p53RE contains a motif which is known to be preferentially targeted by somatic hypermutation. This p53RE is evolutionary conserved in primates, but not in rodents. p53 binds to this RE in vitro and in vivo. Reporter assays revealed that the BCL6 intron 1 can confer p53-dependent transcriptional activation, through the p53RE. BCL6 mRNA and protein levels increased after chemo/radiotherapy in human, but not in murine tissues. The increase in BCL6 mRNA levels was attenuated by the p53 inhibitor PFT-α. Thus, we define the BCL6 gene as a new p53 target. In conjunction with the recent finding that BCL6 represses p53 gene expression (Phan & Dalla-Favera 2004. Nature 432: 635), our data define a novel autoregulatory loop, in which p53 is activated in response to genomic alterations that occur normally during B-cell maturation. In the physiological context, this p53 activation induces BCL6, which down-regulates p53 and spares the cell from p53-regulated apoptosis. This down-regulation of p53 is predicted, in turn, to attenuate BCL6, allowing it to return to baseline levels and thereby defining a tightly controlled time window for completion of B-cell maturation. Disruption of this loop may lead to deregulated BCL6 expression and promote lymphomagenesis.
Up-Regulation of c-Jun contributes to the Induction of Apoptosis by Adaphostin in Human Multiple Myeloma Cells.
Authors: Podar Klaus , Tonon Giovanni , Raab Marc S. , Sattler Martin , Abtahi Dean , Tai Yu-Tzu , LeGouill Steven , Ishitsuka Kenji , Yasui Hiroshi , Depinho Ron A. , Hideshima Teru , Chauhan Dharminder , Anderson Kenneth C.
Published: April 2020

Adaphostin (NCI, Bethesda, MD) has been examined as an alternative to the 2-phenylaminopyrimidine derivate imatinib mesylate, with remarkable efficacy in the treatment of chronic myeloic leukemia (CML) including (1) Bcr/Abl+ cells; (2) imatinib mesylate resistant cells; and (3) Bcr/ Abl - cells. However exact functional mechanisms by which adaphostin induces cell apoptosis are unknown. Previously we have compared effects of adaphostin, a small molecule of the tyrophostin family versus imatinib mesylate in human MM cells: significant concentration - dependent antiproliferative the effects were observed after treatment with adaphostin, but not with imatinib mesylate, on MM cells alone and adherent to BMSCs. Here, we used gene expression microarray to identify signaling molecules modulated by adaphostin. As confirmed by protein expression, several proteins, most prominently c-Jun, were upregulated in a dose- and time- dependent manner. In contrast, c-Jun upregulation was not observed in MM cells treated with imatinib mesylate. Although PARP cleavage was induced, c-Jun upregulation was also not observed after treatment with dexamethasone (0.5 and 1mM), bortezomib (5 and 20nM) or melphalan (5 and 20mM). The functional relevance of adaphostin- induced c-Jun- upregulation was next investigated using small- interfering RNAs targeting c-Jun mRNA. Adaphostin- induced caspase- cleavage is abrogated in MM cells transfected with cJun- specific RNAi, but not with mock-RNAi. Consequently, a decrease of thymidine incorporation induced by adaphostin was only observed in MM cells transfected with mockRNAi, but not c-Jun- specific RNAi. Moreover, our results also demonstrate that adaphostin- induced caspase cleavage is dependent on the expression of c-Jun, but not visa versa. These studies for the first time provide evidence for a specific adaphostin- induced c-Jun upregulation and related induction of apoptosis in MM cells. Based on these findings, ongoing studies are investigating the role of c-Jun in MM pathogenesis.
JS-K, a GST-Activated Nitric Oxide Generator, Induces Apoptosis and Overcomes In Vitro Drug Resistance in Multiple Myeloma Cells.
Authors: Kiziltepe Tanyel , Ishitsuka Kenji , Hideshima Teru , Raje Noopur , Shiraishi Norihiko , Yasui Hiroshi , Catley Laurence , Chauhan Dharminder , Saavedra Joseph E. , Keefer Larry K. , Shami Paul J. , Anderson Kenneth C.
Published: April 2020

Multiple myeloma (MM) is currently an incurable hematological malignancy. A major reason for the failure of currently existing therapies is the chemotherapeutic resistance acquired by the MM cells upon treatment. Overexpression of glutathione S-transferases (GST) has been shown as one possible mechanism of anti-cancer drug resistance in a broad spectrum of tumor cells. JS-K (O2-(2,4-Dinitrophenyl) 1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate) belongs to a class of pro-drugs which are designed to release nitric oxide (NO) on reaction with GST. JS-K can possibly turn GST overexpression to the tumor’s disadvantage by (1) consuming intracellular GSH and preventing drug inactivation; and (2) by exposing tumor cells to high intracellular concentrations of NO. JS-K has potent in vitro and in vivo anti-leukemic activity. The purpose of the present study is to examine the biological effects of JS-K on human MM cells. We demonstrate that JS-K has significant in vitro cytotoxicity on MM cell lines, with an IC50 of 0.3-2 mM at 48 hours. JS-K also induces cytotoxicity on cell lines that are resistant to conventional chemotherapy (i.e., MM1R, RPMI-Dox40, RPMI-LR5, RPMI-MR20). Importantly, no cytotoxic effects of JS-K were detected on peripheral blood mononuclear cells (PBMNC) obtained from healthy volunteers at these doses. Moreover, JS-K could overcome the survival and growth advantages conferred by interleukin-6 (IL-6) and insulin-like growth factor-1 (IGF-1), or by adherence of MM cells to bone marrow stromal cells (BMSC). JS-K caused a transient G2/M arrest followed by apoptosis, as determined by flow cytometric analysis using PI, Annexin V and Apo2.7 staining. JS-K-induced apoptosis was associated with caspase 8, 7, 9 and 3 activation. Interestingly, Fas was upregulated by JS-K, suggesting the involvement of death receptor pathway in induction of apoptosis. JS-K also triggered Mcl-1 cleavage and Bcl-2 phosphorylation, suggesting the involvement of mitochondrial pathway. In addition, apoptosis inducing factor (AIF), endonuclease G (EndoG) and cytochrome c were released into the cytosol during apoptosis. Taken together, these findings suggest the involvement of both intrinsic and extrinsic apoptotic pathways in JS-K-induced apoptosis in MM cells. In summary, our studies demonstrate that JS-K induces apoptosis and overcomes in vitro drug resistance in MM cells. Therefore, JS-K is a novel compound which carries significant potential to be included in the repertoire of existing treatment modalities for MM. Ongoing studies are delineating the mechanism of action of JS-K to provide the preclinical rationale for combination therapies to overcome drug resistance and improve patient outcome.
Acceleration of Leukemogenesis in NUP98-HOXD13 Transgenic Mice by Infection with the MOL4070LTR Virus, and Identification of Collaborating Genes.
Authors: Slape Christopher , Lin Yingwei , Bies Juraj , Wolff Linda , Aplan Peter D.
Published: April 2020

NUP98 is a promiscuous oncogene that is fused to at least sixteen different partner genes in a wide range of haematopoietic malignancies. The NUP98-HOXD13 (NHD13) fusion gene is formed by the t(2;11)(q31;p15), which has been observed in patients with MDS or AML. This fusion gene encodes a protein that fuses the N-terminal portion of NUP98, a nucleoporin involved in mediating RNA and protein transport in and out of the nucleus, with the C-terminal portion of HOXD13, a homeodomain protein not expressed during normal haematopoietic development. We have previously demonstrated that expression of the NHD13 fusion gene in a transgenic mouse model results in an invariably fatal myelodysplastic syndrome (MDS). The median survival for these transgenic mice is 10 months, with no mice surviving beyond 14 months of age. About one-third die due to complications of severe pancytopenia, and about two thirds progress to acute leukemia. To identify genes that might collaborate with the NHD13 transgene, a cohort of thirty mice (15 NHD13 transgenic, 15 normal) were infected with the MOL4070LTR virus at birth and the disease course monitored. All transgenic infected mice developed an acute leukemia, predominantly of the myeloid lineage. The median survival of the transgenic infected mice was only four months, with none surviving beyond 7 months of age. These survival figures are vastly decreased compared to either control group (the wild type infected group or the transgenic uninfected group), suggesting a true synergistic effect between the NUP98-HOXD13 transgene and the genes affected by retroviral insertion events. Cloning of these insertion sites has revealed proximity to numerous genes of interest, including several genes with a known role in haematopoiesis (EpoR, Ifnar2, Gata2, PU.1), development (HOXB8, HOXC9) and/or cancer (p53, Stat5b, Pim1, CyclinD1). In addition two recurrent integration sites were identified near uncharacterized ESTs. Since the NHD13 fusion has been shown to block differentiation, several of the genes we identified (p53, EpoR, Stat5b, Pim1, CyclinD1) fit the evolving AML paradigm that postulates one mutation blocks differentiation, and a second, complementary mutation leads to increased proliferation or decreased apoptosis. Ongoing analysis of these and additional collaborating genes should provide important insights into the process of the MDS to AML transition.
A Genome-Modified Mouse Line with an Insertion Mutation at the AML1/Runx1 Gene Locus Detected from a Patient with Myelodysplastic Syndrome.
Authors: Fukushime-Nakase Yoko , Nakao Mitsushige , Horiike Shigeo , Nishimura Motohiro , Taniwaki Masafumi , Sugimoto Tohru , Okuda Tsukasa
Published: April 2020

AML1 (Acute Myeloid Leukemia 1; also known as Runt-related transcription factor 1: Runx1) encodes the DNA binding subunit of Core-Binding Factor (CBF) transcription factor complex which plays important roles in initiating definitive hematopoiesis, thymocyte development, and platelet production. AML1’s DNA-binding is mediated through its Runt domain, and their further association with the non-DNA-binding subunit, CBFβ stabilizes the resultant ternary complex to be functional. AML1 is frequently involved in leukemia-associated chromosome translocations, through which AML1 fusion-proteins with strong dominant-negative effects to normal CBF are generated, thus contributing to the leukemic transformation. Recently, genomic mutations of the AML1 gene locus have been described to associate with cases of acute myeloblastic leukemia and myelodysplastic syndromes (MDS), and pedigrees of familial platelet disorder with propensity to develop acute myeloblastic leukemia (FPD/AML). Frequent sites of the point-mutations are confined to the residues within the Runt domain, such as, R139, R174, or R177, which are important for the DNA-contact of the molecule. Preliminary results from studies using knockin mice carrying mis-sense mutations for these residues so far revealed that the mutants are biologically inactive when homozygously inherited. However, the mechanisms how the mutations contribute to the onset of the leukemia remain largely to be elucidated. In order to further characterize the aberration of AML1, we generated another genome-modified mouse line which harbors an insertion mutation of this gene locus. As reported previously, we identified a case of refractory anemia with excess of blasts (RAEB) whose blast cells have a mutated allele where three base-pair, ATC, is inserted after codon 150 (I150ins) of the molecules. By this mutation, an additional isoleucine residue is inserted into the 10th β-strand of the Runt domain sequences which provides a facet to interact with CBFβ. We introduced the cDNA of I150ins into AML1/Runx1 gene locus by means of a gene-knockin approach in mouse ES cells, and transmitted the allele into mouse germline through conventional methods. Subsequent heterozygous mice were viable and fertile. In contrast, homozygous mice die in utero similarly to the mice with simple disruption of this gene. These results indicate that this insertion mutation leads to the complete loss of its biologic function as were observed for the point-mutant mice. Further careful examinations of the heterozygous mice for this mutant allele in comparison with those for point-mutation animals and simple haplo-insufficient ones will provide valuable insights into the molecular mechanisms of AML1-related leukemic transformation.
Species-Specific Differences in Intracellular Transport Reveal a Golgi-Localization Requirement for D816V Mutant Kit Receptor To Induce Leukemia.
Authors: Xiang Zhifu , Cain Jennifer , Kreisel Frederike , Tomasson Michael H.
Published: April 2020

Mutations in the c-Kit gene (KIT) that result in constitutive tyrosine kinase activity are associated with mastocytosis/mast cell leukemia, gastrointestinal stromal tumors (GIST), and acute myeloid leukemia (AML). Expression of a murine Kit allele with a mutation in the phosphotransferase domain (Kit D814V) renders murine Ba/F3 cells factor-independent and causes lymphocytic leukemia in mice. An animal model using the homologous human allele (KIT D816V) has not been described. We generated retroviral expression constructs using wild-type and mutant human KIT and murine Kit that achieved equivalent protein levels in human-derived 293T cells. When expressed in murine cells however, despite equivalent transcript levels, the KITD816V protein specifically was present in lower abundance, and unlike the murine mutant, the human D816V was unable to transform Ba/F3 cells without prior selection. In contrast, human KITD816V rapidly transformed human TF-1 cells to factor independence. Fusion of the extracellular and transmembrane domains of murine Kit to the intracellular domain of human KIT (Hybrid KIT WT and Hybrid KITD816V) facilitated higher protein expression in murine cells, and rapid transformation of both Ba/F3 and TF-1 cells. Endoglycosidase digestion revealed that both human and murine mutant Kit receptors were incompletely glycosylated, and by immunohistochemistry we found that the murine and hybrid mutant KIT receptors were predominantly localized to the golgi. However, the human KIT D816V protein, in murine cells, failed to traffic past the endoplasmic reticulum (ER), while in human cells, KITD816V was able to traffic through the ER to the golgi. In murine cells expressing human KITD816V, downstream signaling pathways, including Stat3, Stat5 and Akt were activated to a lesser extent compared to cells expressing the murine KitD814V or hybrid KITD816V. Treatment of cells with small molecule inhibitors of protein trafficking demonstrated that activation of downstream signaling pathways activated by Hybrid KITD816V required golgi but not cell membrane localization. Lastly, mice transplanted with bone marrow transduced with the human KITD816V remained free of disease while mice transplanted with the golgi-localized Hybrid KITD816V succumbed to fatal myeloproliferative disease (MPD) with 100% penetrance. Taken together, our data demonstrate that murine and human cells traffic the KITD816V protein differently and that leukemogenic signals require localization of the mutant receptor to the golgi apparatus. This is the first report of an animal model of myeloid leukemia/MPD using the KIT oncogene and will be useful for elucidating the mechanisms by which KITD816V contributes to leukemogenesis and for testing novel therapeutic strategies.
Deregulation of Retinoic Inducible Gene---Rig-I Underlies the Pathogenesis of Acute Promyelocytic Leukemia.
Authors: Zhu Jiang , Shen Shu-Hong , Chen Li-Juan , Huang Qiu-Hua , Zhang Nan-Nan , Sun Yi-Wu , Emerson Stephen G. , Chen Sai-Juan , Wang Zhu-Gang , Chen Zhu
Published: April 2020

numerous chromosomal translocations, all of which involve the gene encoding retinoid acid receptor a (RARa), resulting in the generation of corresponding oncogenic fusion proteins including PML-RARa and PLZF- RARa (hereafter referred as to X- RARa). However, through deregulating which downstream genetic program(s) X- RARas perturb the hematopoiesis has remained unclear. Here we report that Rig-I, a putative RNA helicase, was identified in RA-induced granulocytic differentiation of APL cell line NB4 cells. Interestingly, the up-regulation of Rig-I was also realized in RA-treated freshly isolated primary APL blasts harboring PML-RARa and HL-60 cell line, but not in RA-treated NB4 variant NB4-R2 (RA refractory) and APL blasts carrying PLZF-RARa, suggesting that the induced Rig-I activity is one essential step underlying the granulocytic differentiation of myeloid leukemia cells. In line with this, the knockout mice of Rig-I produced a phenotype of profound granulocytosis with moderate differentiation blockage; and conversely the overexpression of Rig-I inhibited in vivo hematopoietic reconstitution of transduced normal primitive bone marrow cells. Most importantly, the Rig-I expression was found significantly repressed in myeloid compartment of X-RARa transgenic mice, and that the retroviral transduction of Rig-I cDNA into murine APL blasts carrying hMRP8-PML/RARa greatly decreased their leukemogenesis reconstitution ability in sublethally-irradiated syngeneic recipients. Moreover, we found that Rig-I integrity is required for the full expression of TRAIL, whose expression is critical factor in determining the pool size of myeloid cells. Such, we characterize Rig-I as a crucial negative regulator of granulopoiesis and a potential candidate for developing molecule-based target therapy of myeloid leukemia.