ALBERT

Description: Chronic myelogenous leukemia (CML) is a hematopoietic disorder originating from p210BCR/ABL-transformed stem cells, which begins as indolent chronic phase (CP) but progresses into fatal blast crisis (BC). To investigate molecular mechanism(s) underlying disease evolution, CML-exhibiting p210BCR/ABL transgenic mice were crossed with BXH2 mice that transmit a replication-competent retrovirus. Whereas nontransgenic mice in the BXH2 background exclusively developed acute myeloid leukemia, p210BCR/ABL transgenic littermates developed nonmyeloid leukemias, in which inverse polymerase chain reaction detected 2 common viral integration sites (CISs). Interestingly, one CIS was transgene's own promoter, which up-regulated p210BCR/ABL expression. The other was the 5′ noncoding region of a transcription factor, Zfp423, which induced aberrant Zfp423 expression. The cooperative activities of Zfp423 and p210BCR/ABL were demonstrated as follows: (1) introduction of Zfp423 in p210BCR/ABL transgenic bone marrow (BM) cells increased colony-forming ability, (2) suppression of ZNF423 (human homologue of Zfp423) in ZNF423-expressing, p210BCR/ABL-positive hematopoietic cells retarded cell growth, (3) mice that received a transplant of BM cells transduced with Zfp423 and p210BCR/ABL developed acute leukemia, and (4) expression of ZNF423 was found in human BCR/ABL-positive cell lines and CML BC samples. These results demonstrate that enhanced expression of p210BCR/ABL and deregulated expression of Zfp423/ZNF423 contribute to CML BC.

Description: Chronic immune activation is a major cause for progressive immunodeficiency in human immunodeficiency virus type-1 (HIV) infection. The underlying trigger, however, remains largely unknown. HIV single-stranded RNA is a potent immune activator by triggering Toll-like receptor (TLR) 7/8. Thus, we hypothesized that sustained TLR7 triggering induces chronic immune activation and thereby contributes to progressive immunodeficiency. We used the synthetic compound R848 or a mixture of uridine-rich HIV single-stranded (ss) RNA oligonucleotides—both are potent TLR7/8 agonists—to explore the effects of sustained TLR7 triggering on the murine lymphoid system. Sustained TLR7 triggering induced an immunopathology reminiscent of progressive lymphoid destruction in HIV disease; we observed lymphopenia, elevated proinflammatory cytokines, splenomegaly, contracted lymphoid subsets, and lymphoid microarchitecture alteration with reduced marginal zone B-lymphocytes. Upon exposure to inactivated vesiculo-stomatitis virus, antibody production was abolished, although splenic lymphocytes were activated and total IgG was elevated. Our data imply that HIV itself may directly contribute to immune activation and dysfunction by stimulating TLR7. Thus, manipulation of TLR7 signaling may be a potential strategy to reduce chronic hyper-immune activation and, thereby, disease progression in HIV infection.

Description: The synthetic triterpenoid 2-cyano-3, 12-dioxooleana-1, 9-dien-28-oic acid (CDDO) induces apoptosis in leukemic cells. Here we show that CDDO and its new derivative CDDO-imidazolide (CDDO-Im) trigger apoptosis in multiple myeloma (MM) cells resistant to conventional therapies including melphalan (LR-5), doxorubicin (Dox-40), and dexamethasone (MM.1R, U266, RPMI 8226) without affecting the viability of normal cells. CDDO-IM also triggers apoptosis in bone marrow stromal cells (BMSCs) and decreases interleukin-6 (IL-6) secretion induced by MM cell adhesion to BMSCs. Moreover, CDDO-Im–induced apoptosis in MM cells is not blocked by IL-6 or insulin growth factor-1 (IGF-1). Importantly, CDDO-Im and bortezomib/proteasome inhibitor PS-341 trigger synergistic apoptosis in MM cells associated with loss of mitochondrial membrane potential, superoxide generation, release of mitochondrial proteinscytochrome c/second mitochondria-derived activator of caspases (cyctochrome c/Smac), and activation of caspase-8, -9, and -3. Conversely, the pancaspase inhibitor Z-VAD-fmk abrogates the CDDO-Im + bortezomib–induced apoptosis. Low doses of CDDO-Im and bortezomib overcome the cytoprotective effects of antiapoptotic proteins Bcl2 and heat shock protein-27 (Hsp27) as well as nuclear factor–kappa B (NF-κB)–mediated growth/survival and drug resistance. Finally, combining CDDO-Im and bortezomib induces apoptosis even in bortezomib-resistant MM patient cells. Together, these findings provide the framework for clinical evaluation of CDDO-Im, either alone or in combination with bortezomib, to overcome drug resistance and improve patient outcome in MM. (Blood. 2004;103: 3158-3166)

Description: Prognosis of patients with AML varies widely reflecting the heterogeneity of AML and the background of each patient. It is important for both physicians and patients to predict the response to chemotherapy to correctly select the therapeutic options. In the JALSG-AML97 study, patients were stratified using JALSG scoring system, and those who were categorized into the intermediate or adverse prognosis group were assigned to allo-HSCT during CR1 if they had an HLA-matched sibling. JALSG scoring contains several factors that have 1 or 2 points: 2 points, age 〈 50 years, WBC 50%; 1 point, PS

Description: A randomized study had been performed between December 2001 and December 2005 to assess the optimal post remission therapy for adult AML in the first CR. The updated results are here presented, after a median follow-up of 48 months. JALSG AML201 enrolled 1064 previously untreated AML patients (pts) aged 15–64 yrs. The induction therapy consisted of cytarabine (Ara-C 100 mg/m2 day1–7) and idarubicin (IDR 12 mg/m2 day1–3) (arm A) or cytarabine (100 mg/m2 day1–7) and daunorubicin (DNR 50 mg/m2 day1–5) (arm B). If the patients did not achieve remission after the first induction therapy, then the same therapy was given once more. Pts were categorized into good, intermediate or poor risk groups by risk factors based on the criteria established in previous JALSG AML studies (Miyawaki et al. Cancer 2005). All CR pts were stratified according to the induction, the number of courses of induction, age and karyotype and were randomly assigned to the high dose Ara-C (HDAC) post remission regimen (arm C) or the conventional JALSG post remission regimen (arm D). Arm C: the three courses of HDAC which consisted of Ara-C 2.0 g/m2 q12h day1–5, arm D: the first course consisted of Ara-C 200 mg/m2 day1–5+ mitoxantrone (MIT) 7 mg/m2 day1–3, 2) Ara-C 200 mg/m2 day1–5+ DNR 50 mg/m2 day1–3, 3) Ara-C 200 mg/m2 day1–5+ aclarubicin (ACR) 20 mg/m2 day15, 4) Ara-C 200 mg/m2 day1–5+ etoposide (ETP) 100 mg/m2 day1–5 + vincristine (VCR) 0.8 mg/m2 day 8 + vindesine (VDS) 2 mg/m2 day10. Results: Of the 1064 pts registered, 1057 pts (median age: 47 years) were evaluable. 823 pts (78%) achieved CR after one or two courses of induction therapy. Of the 823 pts in CR, 781 pts were assigned to arm C (n=389) or arm D (n=392). The 5-year OS rate of arm C was 57.8% while that of arm D was 55.9% (p=0.96). The 5-year RFS rate of the CR pts was 42.7% in arm C and 38.9% in arm D (p=0.73). Among the good risk group (n=155), the 5-year OS rate of arm C was 69.9% while that of arm D was 80.5 % (p=0.11), and the 5-year RFS rate of arm C was 54.5% while that of arm D was 55.7% (p=0.53). Among the intermediate risk group (n=439), the 5-year OS rate of arm C was 50.9% while that of arm D was 48.5% (p=0.59), and the 5-year RFS rate of arm C was 41.5% while that of arm D was 36.5% (p=0.50). Among the poor risk group (n=49), the 5-year OS rate of arm C was 12.9% while that of arm D was 17.2% (p=0.58), and the 5-year RFS rate of arm C was 14.3% while that of arm D was 15.5% (p=0.78). In the CBF leukemia group (n=218), the 5-year OS rate of arm C was 75.0% while that of arm D was 65.8% (p=0.17), and the 5-year RFS rate of arm C was 56.5% while that of arm D was 38.7% (p=0.05). Among the young group (=50 yrs) (n=314), the 5-year OS rate of arm C was 51.3% while that of arm D was 40.1% (p=0.16), and the 5-year RFS rate of arm C was 40.0% while that of arm D was 28.1% (p=0.23). After all of consolidation, the lowest WBC count and the duration of neutropenia in arm C were significantly lower and longer than those in arm D. There was a higher rate of documented infection in arm C (20.9%) than in arm D (14.5%) (p〈 0.001). Conclusion: The conventional post remission therapeutic regimen established by JALSG consisting of 4 courses of consolidation was found to be as effective as the three courses of HDAC therapy. HDAC therapy produced a slightly positive effect on RFS in only the CBF leukemia group.

Description: In attempts to isolate myeloid tumor-suppressor genes responsible for 7q deletion, we identified a common microdeletion cluster in chromosome subband 7q21.2 by microarraybased CGH analyses of JMML (ASH Annual Meeting, 2006). This region was also deleted in nearly 30% of unselected adult MDS/AML patients, mostly as a part of monosomy 7 or larger 7q deletions. In this region, there are three poorly-characterized genes (Miki = LOC253012, Kasumi = Samd9, and Titan = Samd9L). Miki encoding a centrosomal protein is likely involved in myelodysplasia and chromosomal instability, which are characteristic of -7/7q- MDS/AML, as is presented in this meeting elsewhere. Kasumi (Samd9) and Titan (Samd9L) are related genes that encode 60% homologous proteins. Neither Kasumi nor Titan has homology with any other proteins or contain known functional motifs. Kasumi and Titan were ubiquitously expressed at a relatively constant level. However, in six cell lines derived from MDS/AML patients harboring monosomy 7, Kasumi protein was barely detectable, whereas Titan expression levels were roughly half of those in other AML cells. The mouse genome contains only Titan and lacks Kasumi gene, suggesting that the function of these two gene products are overlapping. We started to characterize these genes by generating mice deficient in Titan (titan−/−). titan−/− mice appear normal and no hematological abnormalities have been observed, suggesting that additional gene alterations are required for leukemia development. To address this issue, retroviral insertional mutagenesis was applied to the mice. Virus infection induced acute leukemia in homozygous (titan−/−) and heterozygous (titan+/−) mice with higher morbidity and mortality than in wild-type (titan+/+) littermates. Leukemias developed in titan+/+ mice were mainly of T-cell lineage. By contrast, those developed in titan−/− and titan+/− mice were negative for lymphoid markers but expressed various combination of cell surface markers for myeloid (Gr1), monocytic (Mac1), erythyroid (Ter119) and megakaryocytic (CD61) progenitors. Histopathology demonstrated that leukemia cells infiltrated the liver, lung, kidneys and spleen, and a portion of the infiltrated cells were maturated. These data suggests that leukemias that developed in titan-deficient mice represent stem cell malignancy rather than AML. Inverse PCR detected two common integration sites (CIS) specific for titan−/− and titan+/− mice, which induced deregulated expression of a zinc finger transcription factor, Evi1, and a histone demethylase, Fbxl10. In addition, although it was not a CIS, TGFβ was isolated as a major viral integration site in one tumor. These results demonstrated that haploinsufficiency and deficiency of Titan predispose leukemia development through inhibition of TGFβ-mediated signaling or an epigenetic change. Recently, deleterious mutations in the Titan gene were reported to be involved in Normophosphatemic Familial Tumoral Carcinosis, a rare autosomal recessive disease in five families of Jewish-Yemenite origin. Impairment of cell migration is suspected to be a cause of this disease and, indeed, wound healing test revealed that fibroblasts established from titan−/− and titan+/− mice migrate slower than those established from wild-type mice. Relevance of the impairment of cell migration to development of leukemia in titan-deficient mice is currently under investigation.

Description: Between 2001 and 2005, JALSG conducted a randomized study to assess the optimal post remission therapy for adult AML in the first CR. JALSG AML201 enrolled 1064 previously untreated AML patients (pts) aged 15–64 yrs. The induction therapy consisted of cytarabine (Ara-C 100mg/m2 day1–7) and idarubicin (IDR 12mg/m2 day1– 3) (arm A) or cytarabine (100mg/m2 day1–7) and daunorubicin (DNR 50mg/m2 day1– 5) (arm B). If the patients did not achieve remission after the first induction therapy, then the same therapy was given once more. Pts were categorized into good, intermediate or poor risk groups by risk factors based on the criteria established in previous JALSG AML studies. All CR pts were stratified according to the induction, the number of courses of induction, age and karyotype and were randomly assigned to the high dose Ara-C (HDAC) post remission regimen (arm C) or the conventional JALSG post remission regimen (arm D). Arm C: the three courses of HDAC which consisted of Ara-C 2.0g/m2 q12h day1–5, arm D: the first course consisted of Ara-C 200mg/m2 day1–5+ mitoxantrone (MIT) 7mg/m2 day1–3, 2) Ara-C 200mg/m2 day1–5+ DNR 50mg/m2 day1–3, 3) Ara-C 200mg/m2 day1–5+ aclarubicin (ACR) 20mg/m2 day1–5, 4) Ara-C 200mg/m2 day1–5+ etoposide (ETP) 100mg/m2 day1–5 + vincristine (VCR) 0.8mg/m2 day 8 + vindesine (VDS) 2 mg/m2 day10. Results: Of the 1064 pts registered, 1057 pts (median age: 47 years) were evaluable. 825 pts (78%) achieved CR after one or two courses of induction therapy. Of the 825 pts in CR, 781 pts were assigned to arm C or arm D. The 4-year OS rate of arm C was 61.6% while that of arm D was 62.8% (p=0.58). The 4-year RFS rate of the CR patients was 42.8% in arm C and 40.8% in arm D (p=0.65). Among the good risk group, the 4-year OS rate of arm C was 77.0% while that of arm D was 75.8 % (p=0.40), and the 4-year RFS rate of arm C was 54.6% while that of arm D was 53.1% (p=0.71). Among the intermediate risk group, the 4-year OS rate of arm C was 63.2% while that of arm D was 65.7% (p=0.78), and the 4-year RFS rate of arm C was 38.7% while that of arm D was 42.2% (p=0.63). Among the poor risk group, the 4-year OS rate of arm C was 36.3% while that of arm D was 34.1% (p=0.71), and the 4-year RFS rate of arm C was 25.9% while that of arm D was 6.6% (p=0.17). In the CBF leukemia group, the 4-year OS rate of arm C was 79.4% while that of arm D was 66.5% (p=0.09), and the 4-year RFS rate of arm C was 57.7% while that of arm D was 43.7% (p=0.14). Among the young group (=50 yrs), the 4-year OS rate of arm C was 53.4% while that of arm D was 57.7% (p=0.90), and the 4-year RFS rate of arm C was 39.1% while that of arm D was 33.8% (p=0.67). Conclusion: The conventional post remission therapeutic regimen established by JALSG consisting of 4 courses of consolidation was thus found to be as effective as the three courses of HDAC therapy. To further confirm these results, a longer follow-up is therefore needed.

Description: It has been shown that the novel synthetic triterpenoid CDDO inhibits proliferation and induces differentiation and apoptosis in myeloid leukemia cells. In the current study the effects of the C-28 methyl ester of CDDO, CDDO-Me, were analyzed on cell growth and apoptosis of leukemic cell lines and primary acute myelogenous leukemia (AML). CDDO-Me decreased the viability of leukemic cell lines, including multidrug resistant (MDR)-1–overexpressing, p53null HL-60-Dox and of primary AML cells, and it was 3- to 5-fold more active than CDDO. CDDO-Me induced a loss of mitochondrial membrane potential, induction of caspase-3 cleavage, increase in annexin V binding and DNA fragmentation, suggesting the induction of apoptosis. CDDO-Me induced pro-apoptotic Bax protein that preceded caspase activation. Furthermore, CDDO-Me inhibited the activation of ERK1/2, as determined by the inhibition of mitochondrial ERK1/2 phosphorylation, and it blocked Bcl-2 phosphorylation, rendering Bcl-2 less anti-apoptotic. CDDO-Me induced granulo-monocytic differentiation in HL-60 cells and monocytic differentiation in primary cells. Of significance, colony formation of AML progenitors was significantly inhibited in a dose-dependent fashion, whereas normal CD34+ progenitor cells were less affected. Combinations with ATRA or the RXR-specific ligand LG100268 enhanced the effects of CDDO-Me on cell viability and terminal differentiation of myeloid leukemic cell lines. In conclusion, CDDO-Me is an MDR-1– and a p53-independent compound that exerts strong antiproliferative, apoptotic, and differentiating effects in myeloid leukemic cell lines and in primary AML samples when given in submicromolar concentrations. Differential effects of CDDO-Me on leukemic and normal progenitor cells suggest that CDDO-Me has potential as a novel compound in the treatment of hematologic malignancies.

Description: The aim of this study was to investigate the efficacy of allo-SCT as a postremission treatment in patients (pts) with intermediate/poor risk AML in first CR. Previously untreated pts aged15–64 years were eligible. Pts received standard induction therapy consisting of cytarabine (100 mg/m2 d1–7) and idarubicin (12 mg/m2 d1- 3). If the pts did not achieve remission after the first induction therapy, the same induction therapy was given once more. The pts who achieved CR were randomized into an intensified short-course postremission regimen group (arm A) and conventional JALSG postremission regimen group (arm B). Pts were also categorized into good, intermediate or poor risk groups by risk factors based on previous JALSG AML trials using univariate and multivariate analyses. The intermediate or poor risk pts

Description: Abstract 2963 Poster Board II-939 From a common microdeletion cluster located in chromosome subband 7q21.3, we identified three candidate responsible genes (Kasumi=Samd9, Titan=Samd9L and Miki=LOC253012) which encode myeloid-tumor suppressors (BBRC 2009). As we presented previously (ASH annual meeting, 2008), Miki encodes a centrosomal protein and is involved in myelodysplasia and chromosomal instability. On the other hand, Kasumi and Titan, that encode 60% identical proteins, are poorly characterized. Recent reports revealed that bi-allelic point mutations in Kasumi gene cause a rare fatal skin disease, Normophosphatemic Familial Tumoral Calcinosis (NFTC). To identify the contributions of these genes to leukemogenesis, we initially generated Titan deficient mice (mouse genome contains only Titan and lacks Kasumi gene). Although heterozygous (titan+/−) and homozygous (titan-/-) mice were born and grown normally without hematological abnormalities, they naturally developed AML at high frequency after they reached 20 months old. This unusually long latency suggests that additional gene alterations are required for leukemia development. Thus we attempted to accelerate the onset of leukemia by retroviral insertional mutagenesis. Virus infection induced various myeloid leukemias after 10 to 12 months in almost all titan+/− and titan-/- mice. Inverse PCR detected two common virus integration sites specific for titan+/− and titan-/- mice, which induced deregulated expression of a zinc finger transcription factor, Evi1, and a histone H3K36 demethylase, Fbxl10. We next performed mouse BMT using titan-/- and +/+ bone marrow cells transduced with Evi1 retrovirally. Mice transplanted with titan(+/+)/Evi1 overexpressing cells developed MDS or AML after 7 months. By contrast, most mice transplanted with titan(-/-)/Evi1 overexpressing cells developed AML within 6 months after BMT, confirming co-operation between loss of Titan and Evi1 overexpression in myeloid leukemogenesis. Both Evi1 and Fbxl10 are reported to downregulate p15Ink4b tumor suppressor gene. Moreover, in human secondary MDS, DNA methylation in the promoter region of p15Ink4b is closely associated with 7q deletion. Thus we compared Fbxl10 and p15Ink4b expressions between AML/MDS samples with or without 7q deletion. We found that 7q deletion was correlated with higher Fbxl10 and with lower p15Ink4b levels, suggesting that silencing of p15ink4b through transcriptional and epigenetical mechanisms would be involved in leukemia with 7q deletion. To elucidate the function of Kasumi and Titan, we firstly immunoprecipitated Titan binding proteins from FLAG-Titan expressing cells and identified two specific bands around 150 and 70KDa. Mass spectrometry analysis showed that they correspond to Flightless1 (Fli1) and Scinderin (Scin), respectively. Because both Fli1 and Scin belong to the gelsolin superfamily proteins that bind to and sever actin filaments, we speculated that Titan is involved in cell movement via actin remodeling. To analyze this, we established Titan-knockdown (K/D) mouse fibloblasts by introducing Titan-specific shRNA-expressing vectors and observed their migration under time-lapse microscopy. In wound-healing assay, Titan-K/D cells migrated slower towards wound edge with loss of polarity. Each cell moved restlessly by quickly changing the direction. In these cells, lamellipodial protrusions rapidly formed and retracted. In accordance with this phenotype, activity of Rac1, a Rho GTPase, increased in Titan-K/D cells. This abnormality in cell migration is likely involved in pathogenesis of NFTC, i.e., severe inflammations in skin and mucosae. Moreover, since recent reports revealed the contribution of hyperactivated Rac1 to transformation of hematopoietic stem cells through abnormal actin remodeling, our findings suggest that deletion of Kasumi or Titan is a cue to cause AML/MDS through aberrant Rac1 activation. Disclosures: No relevant conflicts of interest to declare.