ALBERT

Description: Introduction: Although recent studies have refined the classification of B-progenitor and T-lineage acute lymphoblastic leukemia into gene-expression based subgroups, a comprehensive integration of significantly mutated genes and pathways for each subgroup is needed to understand disease etiology. Methods: We studied 2789 children, adolescents and young adults (AYA) with newly diagnosed B-ALL (n=2,322 cases) or T-ALL (n=467) treated on Children's Oncology Group (n=1,872) and St. Jude Children's Research Hospital trials (n=917). The cohort comprised childhood NCI standard-risk (41.8%; age range 1-9.99 yrs, WBC ≤ 50,000/ml), childhood NCI high-risk (44.5%; age range ≥10 to 15.99 yrs) and AYA (9.9%; age range 16-30.7 yrs). Genomic analysis was performed on tumor and matched-remission samples using whole transcriptome sequencing (RNA-seq; tumor only; n=1,922), whole exome sequencing (n=1,659), whole genome sequencing (n=757), and single nucleotide polymorphism array (n=1,909). Results: For B-ALL, 2104 cases (90.6%) were classified into 26 subgroups based on RNA-seq gene expression data and aneuploidy or other gross chromosomal abnormalities (iAMP21, Down syndrome, dicentric), deregulation of known transcription factors by rearrangement or mutation (PAX5 P80R, IKZF1 N159Y), or activation of kinase alterations (Ph+, Ph-like). For T-ALL, cases were classified into 9 previously described subtypes based on dysregulation of transcription factor genes and gene expression. In 1,659 cases subject to exome sequencing (1259 B-ALL, 405 T-ALL) we identified 18,954 nonsynonymous single nucleotide variants (SNV) and 2,329 insertion-deletion mutations (indels) in 8,985 genes. Overall, 161 potential driver genes were identified by the mutation-significance detection tool MutSigCV or by presence of pathogenic variants in known cancer genes. Integration of sequence mutations and DNA copy number alteration data in B-ALL identified 7 recurrently mutated pathways: transcriptional regulation (40.6%), cell cycle and tumor suppression (38.0%), B-cell development (34.5%), epigenetic regulation (24.7%), Ras signaling (33.0%), JAK-STAT signaling (12.0%) and protein modification (ubiquitination or SUMOylation, 5.0%). The top 10 genes altered by deletion or mutation in B-ALL were CDKN2A/B (30.1%), ETV6 (27.0%), PAX5 (24.6%), CDKN1B (20.3%), IKZF1 (17.6%), KRAS (16.5%), NRAS (14.6%), BTG1 (7.5%) histone genes on chromosome 6 (6.9%) and FLT3 (6.1%), and for T-ALL, CDKN2A/B (74.7%), NOTCH1 (68.2%), FBXW7 (21.3%), PTEN (20.5%) and PHF6 (18.2%) (Figure 1A). We identified 17 putative novel driver genes involved in ubiquitination (UBE2D3, UBE2A, UHRF1, and USP1), SUMOylation (SAE1, UBE2I), transcriptional regulation (ZMYM2, HMGB1), immune function (B2M), migration (CXCR4), epigenetic regulation (DOT1L) and mitochondrial function (LETM1). We also observed variation in the frequency of genes and pathways altered across B-ALL subtypes (Figure 1B). Interestingly, alteration of SAE1 and UBA2, novel genes that form a heterodimeric complex important for SUMOylation, and UHRF1 were enriched in ETV6-RUNX1 cases. Deletions of LETM1, ZMYM2 and CHD4 were associated with near haploid and low hypodiploid cases. Deletion of histone genes on chromosome 6 and alterations of HDAC7 were enriched in Ph+ and Ph-like ALL. Mutations in the RNA-binding protein ZFP36L2 were observed in PAX5alt, DUX4 and MEF2D subgroups. Genomic subtypes were prognostic. ETV6-RUNX1, hyperdiploid, DUX4 and ZNF384 ALL were associated with good outcome (5-yr EFS 91.1%, 87.2%, 91.9% and 85.7%, respectively), ETV6-RUNX1-like, iAMP21, low hyperdiploid, PAX5 P80R and PAX5alt were associated with intermediate outcome (5-yr EFS 68.6%, 72.2%, 70.8%, 77.0% and 70.9%, respectively), whilst KMT2A, MEF2D, Ph-like CRLF2 and Ph-like other conferred a poor prognosis (55.5%, 67.1%, 51.5% and 62.1%, respectively). TCF3-HLF and near haploid had the worst outcome with 5-yr EFS rates of 27.3% and 47.2%, respectively. Conclusions: These findings provide a comprehensive landscape of genomic alterations in childhood ALL. The associations of mutations with ALL subtypes highlights the need for specific patterns of cooperating mutations in the development of leukemia, which may help identify vulnerabilities for therapy intervention. Disclosures Gastier-Foster: Bristol Myers Squibb (BMS): Other: Commercial Research; Incyte Corporation: Other: Commercial Research. Willman:to come: Patents & Royalties; to come: Membership on an entity's Board of Directors or advisory committees; to come: Research Funding. Raetz:Pfizer: Research Funding. Borowitz:Beckman Coulter: Honoraria. Zweidler-McKay:ImmunoGen: Employment. Angiolillo:Servier Pharmaceuticals: Consultancy. Relling:Servier Pharmaceuticals: Research Funding. Hunger:Jazz: Honoraria; Amgen: Consultancy, Equity Ownership; Bristol Myers Squibb: Consultancy; Novartis: Consultancy. Loh:Medisix Therapeutics, Inc.: Membership on an entity's Board of Directors or advisory committees. Mullighan:Amgen: Honoraria, Other: speaker, sponsored travel; Loxo Oncology: Research Funding; AbbVie: Research Funding; Pfizer: Honoraria, Other: speaker, sponsored travel, Research Funding; Illumina: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: sponsored travel.

Description: Abstract 2839 Poster Board II-815 Multiple myeloma (MM) remains largely incurable. The existence of the putative myeloma stem cell population may account for drug-resistance and relapse. Previous investigation showed that the CD138-negative (CD138-) MM cell fraction contains the putative MM stem cells. However, little is known about the molecular characteristics of MM stem cells, which makes it difficult to specifically target such cells. In this study, by comparing gene expression profiles (GEP) of CD138+ and CD138- cells isolated from 10 MM cell lines, we discovered that RARα is the top one over-expressed gene in CD138- MM stem cells. RARα has two major isoforms, RARα1 and RARalpha2. Real-time PCR detected significantly higher expression of RARalpha2 but not of RARalpha1 in CD138- MM cells compared with CD138+ cells. We recently reported that increased RARalpha2 expression at diagnosis resulted in a significantly shorter overall-survival (P = 0.003); importantly, ATRA selectively killed RARalpha2-positive myeloma cells, but not RARalpha2-negative cells. These results suggested that ATRA could be used to eradicate specifically RARalpha2-over-expressing MM stem cells. Indeed, we found that ATRA selectively killed CD138- MM stem cells (P 〈 0.01) from ARP-1 and OCI-MY5 human myeloma cell lines and 5T33 murine myeloma cells but spared the CD138+ tumor cells from these cell lines. WNT and Hedgehog (HH) signaling pathways were activated in CD138- stem cells. To our surprise, ATRA treatment (10-6M) further increased WNT and HH signaling pathways in CD138- myeloma cells, based on increased protein levels of β-catenin and cleaved Shh/Ihh in ATRA-treated cells. Stimulation of these signaling pathways by LiCl (5 mM) and/or SHH(20 μg/mL) partially abrogated ATRA-induced cytotoxities on CD138- MM cells, demonstrating that stimulation of WNT and HH signaling induced partial ATRA-resistance in CD138- cells. A combinational treatment of ATRA (1 μM), a COX-2 inhibitor celecoxib (shown to inhibit WNT signaling, 50 μM) and a HH signaling inhibitor cyclopamine (10 μM) induced synergistic effects on cell death and growth inhibition of CD138- and RARalpha2-over-expressing MM cells in-vitro. In the 5T33 murine myeloma model, the combination of ATRA (20mg/Kg), celecoxib (10mg/Kg) and cyclopamine (20mg/Kg) induced synergistic inhibition of tumor growth in-vivo. Thus, our study provides novel approaches to specifically target MM stem cells. Disclosures: No relevant conflicts of interest to declare.

Description: Specific genetic alterations in multiple myeloma (MM) may cause more aggressive diseases. Paired gene array analysis on 51 samples showed that retinoic acid (RA) receptor α (RARα) expression significantly increased at relapse compared with diagnosis. RARα encodes 2 major isoforms: RARα1 and RARα2. In this study, we examined the function of RARα2 in MM. Reverse transcription–polymerase chain reaction (RT-PCR) revealed ubiquitous RARα1 expression in MM cells, but RARα2 was expressed in 26 (32%) of 80 newly diagnosed patients and 10 (28%) of 36 MM cell lines. Patients with RARα2 expression had a significantly shorter overall survival on identical treatments. The presence of RARα2 remained significant on multivariate analysis. Knockdown of RARα2 but not RARα1 induced significant MM cell death and growth inhibition, and overexpressing RARα2 activated STAT3 and mitogen-activated protein kinase kinase (MEK)/extracellular signal–regulated kinase (ERK) signaling pathways. Interestingly, all-trans retinoic acid (ATRA) treatment induced potent cell death and growth inhibition in RARα2+ but not RARα2− MM cells; overexpressing RARα2 in RARα2-deficient MM cells restored sensitivity to ATRA. Furthermore, ATRA treatment significantly inhibited the growth of RARα2-overexpressing MM tumors in severe combined immunodeficiency (SCID) mouse model. These findings provide a rationale for RA-based therapy in aggressive RARα2+ MM.

Description: Pediatric cancers are distinct from adult cancers in both their genomic alterations and therapeutic responses. Fms-like tyrosine kinase 3 (FLT3) mutations, especially internal tandem duplications (ITD), are among the most common mutations in acute myeloid leukemia (AML). FLT3-ITD mutations occur in approximately 15% of pediatric and 25-30% of adult AML, and are generally associated with poor prognosis. However, a number of studies have suggested that FLT3-ITD-positive(+) AML requires additional cooperative mutations. The objective of this study was to characterize the mutational landscape in a cohort of FLT3-ITD+ pediatric AML patients (median age,12.6 years; range, 2.8-19.2 years) enrolled to the AML02 and AML08 trials using samples obtained at diagnosis (n=34) and paired diagnosis/relapse samples (n=5). Children with promyelocytic leukemia were excluded. Samples were analyzed by RNASeq, a targeted 95 gene next generation sequencing (NGS) panel, and whole exome sequencing (WES). At diagnosis, 58.8% of the samples contained fusion genes; 41.2% were NUP98-NSD1, 11.8% were novel fusions (NSD1-CAPRIN1, NSD1-RALBP1, RUNX1-BCL11B, ZEB2-BCL11B), and 5.9% were previously reported fusions (CBFB-MYH11, DEK-NUP214). The NGS panel identified that WT1 and NPM1 were routinely mutated at a frequency of 32.4% and 20.6% respectively. While the NPM1 mutation was either a 4bp insertion at amino acid (a.a.) 287 or 288, WT1 mutations were heterogenenous with missense mutations, insertions and deletions all being reported. WT1 mutations and NUP98-NSD1 co-associated in 7 patients, 1 patient also harbored a TYK2 mutation; in the remaining 7 patients with NUP98-NSD1 fusions, a mutation in RAD21 or NRAS was observed in 2 patients. For samples with other fusions (n=6), we detected an average of 1 additional mutation per sample, which included mutations (variant allele frequency; VAF) in DNMT3A (0.44), IDH2 (0.49), KIT (0.37), NPM1 (0.51), PLCG2 (0.44), RAD21 (0.55), and SMC1A (0.47). No fusion genes were observed in 13 patients. In this latter subset, mutations in NPM1 (n=6) and WT1 (n=3) were observed. Other alterations that were identified in these samples included mutations in DNMT3A, IDH2, PLCG2, and PRKCB, which co-occurred with NPM1 mutations. Three patients did not harbor a fusion gene or a gene mutation by our analysis. When looking at cumulative incidence of relapse or resistant disease, our study results are concordant with previous reports where a NUP98-NSD1 fusion associated with worse prognosis (hazard ratio [HR] = 3.2, p = 0.02), but FLT3-ITD allelic ratio 〉0.4 was not prognostic (HR = 1.1, p=0.87). NPM1 mutations were not significantly associated with better prognosis (HR = 0.2, p = 0.11). We next sought to identify relapse specific alterations by analysis of paired diagnosis/relapse samples by RNASeq, NGS panel, and WES. Notably, the FLT3-ITD mutation was maintained at relapse in all samples. From the NGS panel, we observed the emergence of a MED12 mutation (P1751Q, VAF 0.37) and WT1 mutation (p.S152*, VAF 0.19) at relapse; a mutational switch in WT1 from diagnosis to relapse was also observed (5bp insertion at a.a. 157 to 2bp insertion at a.a. 158). By RNASeq analysis, we found a novel relapse specific fusion gene, LUZP6-OSBL1A. From exome sequencing, mutations in transcription factors were observed at relapse such as CREBBP, GLI3, and TBX20. Our analysis of relapse specific genes showed recurrent mutations in HUWE1, OGT, NACAD, and UNC13A. Intriguingly, both OGT and HUWE1 have been implicated in cancer metabolic reprogramming, and regulate MYC transcriptional programs. OGT is an O-Linked β-N-acetylglucosamine (O-GlcNAc) transferase involved post-translational modification of serine and threonine residues. HUWE1 is an E3 ubiquitin ligase that has been established as a tumor suppressor and previously reported to be mutated in AML. In conclusion, we demonstrate that additional genomic alterations are observed in the majority of pediatric FLT3-ITD+ AML samples evaluated, with a high proportion of samples containing fusion genes, WT1 and NPM1 mutations. We also identified novel fusion genes and mutations that have not been previously reported in pediatric FLT3-ITD+ AML, including relapse specific mutations. These results provide further biological insight into the genomic heterogeneity of pediatric FLT3-ITD+ AML, warranting further investigations in larger patient cohorts. Disclosures Inaba: Arog: Research Funding.

Description: Hepatic veno-occlusive disease (VOD) is a common complication of hematopoietic cell transplantation (HCT), especially patients with β-thalassemia major (TM). To estimate whether incidence of VOD recently decreased after using NF-08-TM protocol (NP), 311 TM-HCTs performed from February 2003 to June 2013 were analyzed. 241 patients received NP in or after 2009 and 70 received non-NP before 2009. VOD was diagnosed by Seattle criteria (SC) or Baltimore Criteria (BC). Patients were stratified by Nanfang (NF) criteria. A total of 31(10.0%) and 14 (4.5%) HCTs developed VOD (6 and 5 developed severe VOD) in SC and BC cohorts, respectively. The incidence of VOD was significantly lower in NP versus non-NP groups and in Class 2 versus Class 3 groups. Overall survival was significantly higher in NP versus non-NP cohorts. Rate of VOD in alternative donor transplant (ADT) was similar to that in matched sibling transplant (MST). Requirement of platelet and value of D-dimer significantly increased in the VOD patients. Our study showed the incidence of VOD significantly decreased in our center after using NP. ADT was similar to MST on rate of VOD. NF criteria of stratification can indicate occurrence of VOD. The SC can be more suitable criteria for early diagnosis. Disclosures No relevant conflicts of interest to declare.

Description: Background: Mixed phenotype acute leukemia (MPAL) is a high risk leukemia with features of acute myeloid (AML) and acute lymphoblastic leukemia (ALL), either due to co-expression of antigens of multiple lineages, or the presence of multiple immunophenotypically distinct populations. WHO 2008 classifies MPAL as T/myeloid (T/M), B/myeloid (B/M), MLL rearranged (MLL) MPAL, BCR-ABL1 (Ph+) MPAL, and MPAL not otherwise specified (NOS). Patients are managed with divergent chemotherapeutic approaches with survival estimates of 50-70%. Apart from Ph+ and MLL rearrangement, the genetic basis of MPAL is poorly defined. Our goal was to define the molecular basis of MPAL, and to compare with potentially related forms of leukemia (AML, T-ALL and early T-cell precursor (ETP) ALL) as a rational foundation for future trials. Furthermore, we examined whether multi-lineal cases harbor genetically distinct subclones, or arise from the acquisition of founding alterations in a multi-lineage hematopoietic progenitor. Methods: 155 cases of pediatric leukemia initially diagnosed as MPAL were studied by central pathology review and/or central flow cytometry (134 cases), confirming the diagnosis according to WHO criteria in 115 cases (fig. 1). Median age was 7 years (0-18) with 52 T/M, 37 B/M, 15 MLL, 8 NOS, and 2 Ph+ (fig. 2). Samples were studied by whole genome and/or exome, RNA sequencing, and SNP array analysis. 44 multi-lineal samples were flow sorted into 2-4 lymphoid, myeloid, and ambiguous subpopulations (15 T/M, 19 B/M, 7 MLL, 1 Ph+, 2 NOS) and subjected to exome sequencing and SNP array. Mutational data were compared to data from 196 AML, 39 ETP-ALL, and 245 T-ALL cases. Results: We identified 35 recurrently mutated genes, the most common of which were WT1 (21%), FLT3 (18%), NRAS (16%), JAK3 (11%), RUNX1 (11%), KMT2D (9%), PTPN11 (9%), ASXL1 (7%), and CREBBP (7%). T/M and B/M subtypes are characterized by distinct patterns of genomic alteration. 48% of T/M cases harbored in-frame chimeric fusion, several of which are described in T-ALL, including ETV6-NCOA2 and ZEB2-BCL11B, NUP214-ABL1 and PICALM-MLLT10, and novel fusions involving hematopoietic regulators (e.g. ETV6-MAML and MNX1-IKZF1). 42% of B/M cases had in-frame fusions of ZNF384 with CREBBP, EP300, and TCF3, while we also identified isolated fusions involving ERG and NF1. Mutations of Ras signaling genes were present in 50% of B/M cases, in contrast to 10% of T/M cases. Epigenetic modifying genes, including CREBBP, SETD2, KMT2D, EZH2 and SUZ12 were mutated in 45% of the combined T/M and B/M cohorts. Cases with MLL gene rearrangements had few sequence alterations. In comparison to other subtypes of leukemia, the mutational spectrum of T/M MPAL, with alterations in transcription factors (60% cases), epigenetic genes (50%) and JAK-STAT signaling (35%) was more similar to ETP-ALL (64%, 72%, 44%) and T-ALL (49%, 60%, 21%) than to AML (19%, 21%, 11%). Similarly, B/M cases have increased alterations in these pathways (42%, 42%, 25%) compared to AML. Sequencing of MPAL subpopulations revealed that 27% of cases had the same SNVs/indels in each subpopulation, and 47% of cases had at least two-thirds of mutations present in each subpopulation. All multi-lineal cases with alterations of regulators WT1 and RUNX1 showed similar allele frequencies of these mutations in all populations. Alternatively, cases with mutations in signaling (FLT3, NRAS, KRAS, PTPN11) or epigenetic regulatory genes (CREBBP, KMT2D, SETD2) only showed consistent presence of alterations across each subpopulation in 60% of the cases. Conclusions: Our analysis has shown that T/M and B/M MPAL are distinct subtypes of leukemia. B/M MPAL is characterized by frequent RAS pathway mutations and ZNF384 fusions with multiple different fusion partners, suggesting that this gene plays a critical role in hematopoietic development for progenitor cells with B lymphoid and myeloid potential. The findings of mutational similarity to ETP ALL, and sharing of genomic lesions between subclones in the majority of cases strongly suggests that MPAL represents part of a spectrum of immature leukemias that arise in a hematopoietic progenitors that may propagate multiple immunophenotypic populations. These results will guide the design of therapeutic strategies for each subtype of MPAL and ETP ALL, and xenografts representative of each subtype are being used to examine sensitivity to therapeutic agents. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures Loh: Abbvie: Research Funding; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees. Zwaan:Pfizer: Research Funding; Pfizer: Consultancy. Reinhardt:Pfizer: Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees; Boehringer Ingelheim: Membership on an entity's Board of Directors or advisory committees; Jazz Pharma: Other: Travel Accomodation. Inaba:Arog: Research Funding. Mullighan:Loxo Oncology: Research Funding; Incyte: Membership on an entity's Board of Directors or advisory committees; Amgen: Speakers Bureau.

Description: Abstract 2734 Poster Board II-710 We have previously demonstrated that parthenolide (PTL), a naturally occurring small molecule found in feverfew Chrysanthemum parthenium, induces apoptosis in primary acute myeloid leukemia (AML) cells, including the stem and progenitor cell compartment. Based on these preclinical findings, a PTL derivative (dimethylamino parthenolide) is currently being evaluated in a phase I clinical trial. However, despite the promising activity of PTL, its underlying mechanism of action remains poorly understood. Thus, we have undertaken biochemical studies to better characterize how PTL mediates leukemia-specific cell death. Chemically, the key structural feature of PTL is its alpha-metheylene-gamma-lactone moiety, which via Michael reaction is predicted to mediate potent free thiol scavenging, an activity readily observed in PTL-treated cells. Reported consequences of PTL chemical reactivity in a variety of cell types, represent a broad range of activities that include inhibition of NFkB, activation of p53, ubiquitination of MDM2, inhibition of DNMT1 and inhibition of HDAC1. To better define the specific activities responsible for parthenolide-mediated leukemia cell death, we have employed two general approaches. First, we generated a biotinylated analog of parthenolide (PTL-biotin), which was shown to retain the anti-leukemia activity of the parent compound. PTL-biotin was then used in biochemical pull-down assays to purify parthenolide target proteins, followed by liquid phase chromatography-mass spectrometry (LC-MS) for protein identification. To further verify molecular interactions, native non-biotinylated parthenolide was used to compete binding between candidate targets and PTL-biotin. These studies identified HSP70 as a direct target of PTL. Notably, cysteine-17 of HSP70 is exposed to the ADP/ATP binding site crevice and a molecular docking study indicates that the covalent attachment of PTL to this residue should disrupt the ATP hydrolysis function of the protein. These findings imply that inhibition of HSP70 may contribute to the cell death mechanism underlying PTL anti-leukemia based activity. As a second approach to characterizing PTL, we have performed comparative studies using the closely related compound costunolide (CSN). Since previous structure-activity studies with PTL analogs revealed that opening of the epoxide ring at C4-C5 of the molecule completely destroys the anti-tumor activity, we sought to utilize a compound lacking this feature. CSN lacks the epoxide group, but is otherwise identical to PTL, and retains the key alpha-metheylene-gamma-lactone moiety. Interestingly, at concentrations where PTL is highly cytotoxic, CSN does not induce leukemia-specific cell death (less than 10% death for primary AML cells at 7.5 microM). Analysis of CSN activity demonstrated that despite the lack of AML cell death, CSN still induced loss of free thiols and increased reactive oxygen species in a fashion comparable to PTL (as measured by mBBR and CM-H2DCFDA based flow cytometry). However, CSN is markedly less effective as an inhibitor of NFkB activity (measured by phosphorylation level of NFkB p65). Taken together these findings indicate that oxidative stress alone is not sufficient for PTL-mediated cell death, and further extend previous molecular genetic data demonstrating that NFkB inhibition is an important component of the overall cell death mechanism. The data also show that the alpha-metheylene-gamma-lactone moiety alone is not sufficient to mediate all aspects of PTL activity, and that at least some activity/specificity is created by juxtaposition of the epoxide group. Based on these studies, as well as previous data, we propose that inhibition of NFkB and HSP70 are components of the parthenolide-mediated cell death mechanism, and that oxidative stress is a necessary but not sufficient aspect of its leukemia-specific activity. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2809 Poster Board II-785 We recently observed that ATRA treatment selectively kills RARalpha2-expressing, while sparing RARalpha2-deficient MM cells. Previous investigations in the colon cancer cells have shown that ATRA inhibits WNT signaling through down-regulation of COX-2. Therefore, we wanted to evaluate the role of WNT signaling in ATRA-induced cell death and growth inhibition of RARalpha2-expressing myeloma cells. To our surprise, we found that ATRA treatment activated but not inhibited WNT signaling in RARalpha2-expressing myeloma cells, based on increased β-catenin levels in ATRA-treated cells. ATRA exerted minimal effects on activation of WNT signaling pathway in RARalpha2-deficient MM cells, and forced expression of RARalpha2 in RARalpha2-deficient cells restored the stimulatory activities of ATRA on the WNT signaling pathway, demonstrating that RARalpha2 expression is required for the ATRA-induced stimulation of WNT signaling in MM cells. Lithium chloride (LiCl) treatment, which activates WNT signaling, partially abrogated ATRA-induced cell death and growth inhibition in RARalpha2-expressing cells, indicating that ATRA-induced activation of WNT signaling resulted in ATRA-resistance and decreased killing of MM cells, suggesting that a combination of targeting WNT signaling pathway and ATRA treatment is necessary for ATRA-based therapy of RARalpha2-expressing myeloma. COX-2 inhibition blocks WNT signaling in colon cancer. Similarly, we found that a COX-2 inhibitor CAY10404 also blocked WNT signaling in RARalpha2-expressing cells as well as in ATRA-treated cells. Interestingly, CAY10404 activated MEK/ERK signaling pathway, while ATRA abrogated CAY10404-induced activation of MEK/ERK signaling pathway. These results demonstrate that the combination of ATRA and COX-2 inhibitor exerts synergistic inhibitory effects on both WNT and MEK/ERK signaling pathways. A combination of ATRA and the COX-2 inhibitor resulted in synergistic cytotoxicity of RARalpha2-expressing MM cells in-vitro. More importantly, the combination of ATRA and CAY10404 also resulted in a synergistic growth inhibition of established MM tumors in SCID mice. Our study demonstrates the importance of targeting WNT signaling in ATRA-based therapy in RARalpha2-expressing myeloma and provides a rationale for the combinational use of ATRA and COX-2 inhibitors. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 126 We previously reported that CKS1B may influence myeloma (MM) cell growth and survival through SKP2/p27Kip1-dependent and -independent mechanisms. However, there is still no direct evidence to prove that CKS1B has a role in MM cell proliferation and disease progression. The present study was performed to establish its functional role and define CKS1B-mediated SKP2/p27Kip1-independent down-stream signaling pathways. CKS1B was over-expressed in OCI-MY5 and XG1 MM cell lines by lentivirus. Western blots confirmed CKS1B over-expression. Cells were cultured in medium containing 1% fetal bovine serum for 7 days. CKS1B-transfection resulted in increased cell proliferation compared to empty-vector (EV)-transfected controls. We also examined the role of CKS1B in myeloma resistance to the general used chemotherapeutic drugs, such as bortezomib (5nM), doxorubicin (100nM) and etoposide (100nM). Untreated cells and empty-vector (EV)-transfected cells with or without drug treatments served as controls. Significant less inhibition of cell growth and cell death was observed after drug treatment in CKS1B-transfected cells compared with controls (P 〈 .05). To screen down-stream signaling pathways associated with cell growth and survival in OCI-MY5, MS28PE and XG-1 cells were transfected with specific CKS1B-shRNA, which resulted in decreased phosphorylation of MEK1/2, ERK1/2, STAT3, MCL1 and BCL2 compared to wild-type and control cells, transfected with scrambled CKS1B-shRNA. To confirm these results, we examined the alteration of STAT3, MEK/ERK and BCL2 signaling pathways in OCI-MY5 and XG1 cells after forced over-expression of CKS1B. Increased levels of p-MEK1/2, p-ERK1/2, p-STAT3, MCL1 and p-BCL2 were observed compared to the EV-transfected controls, confirming that CKS1B activates STAT3, MEK/ERK and BCL2 signaling pathways. In Contrast, SKP2 over-expression or p27Kip1 inhibition resulted in inhibition of STAT3 and MEK/ERK pathways with no remarkable changes inBCL2. Further investigation showed that BCL2 is a downstream target of MEK/ERK signaling. Stimulation of STAT3, MEK/ERK and BCL2 signaling pathways only partially abrogated MM cell death and growth inhibition induced by CKS1B knockdown. Targeting either the STAT3, MEK/ERK or BCL2 signaling pathway with specific inhibitors induced significant MM cell death and growth inhibition in CKS1B-over-expressing MM cells; their combination had a synergistic effect on cell death and growth inhibition. Our findings provide a rationale for targeting STAT3 and MEK/ERK/BCL2 signaling in the therapy of aggressive CKS1B-overexpressing MM, which shows increased proliferation and drug-resistance Disclosures: No relevant conflicts of interest to declare.

Description: We used a novel NF-08-TM transplant protocol based on intravenous busulfan, cyclophosphamide, fludarabine, and thiotepa in 82 consecutive patients with β-thalassemia major (TM), including 52 with allogeneic peripheral blood stem cell transplantation (PBSCT) from unrelated donors (UDs) with well-matched human leukocyte antigens and 30 with hematopoietic stem cell transplantation (HSCT) from matched sibling donors (MSDs). The median age at transplantation was 6.0 years (range, 0.6-15.0 years), and the ratio of male-to-female patients was 56:26. The median follow-up time was 24 months (range, 12-39 months). The estimated 3-year overall survival and TM-free survival were 92.3% and 90.4% in the UD-PBSCT group and 90.0% and 83.3% in the MSD-HSCT group. The cumulative incidences of graft rejection and grades III-IV acute graft-versus-host disease were 1.9% and 9.6%, respectively, in the UD-PBSCT group and 6.9% and 3.6%, respectively, in the MSD-HSCT group. The cumulative incidence of transplant-related mortality was 7.7% in the UD-PBSCT group and 10.0% in the MSD-HSCT group. In conclusion, UD-PBSCTs using the well-tolerated NF-08-TM protocol show similar results to MSD-HSCTs and can be used to treat β-thalassemia patients in the absence of MSDs.