ALBERT

Description: Ceramide is a lipid second messenger derived from the hydrolysis of sphingomyelin by sphingomyelinases (SMases) and implicated in diverse cellular responses, including growth arrest, differentiation, and apoptosis. Defects in the neutral SMase (nSMase) gene Smpd3, the primary regulator of ceramide biosynthesis, are responsible for developmental defects of bone; regulation of ceramide levels have been implicated in macrophage differentiation, but this pathway has not been directly implicated in human cancer. In a genomic screen for gene copy losses contributing to tumorigenesis in a mouse osteosarcoma model, we identified a somatic homozygous deletion specifically targeting Smpd3. Reconstitution of SMPD3 expression in mouse tumor cells lacking the endogenous gene enhanced tumor necrosis factor (TNF)–induced reduction of cell viability. Nucleotide sequencing of the highly conserved SMPD3 gene in a large panel of human cancers revealed mutations in 5 (5%) of 92 acute myeloid leukemias (AMLs) and 8 (6%) of 131 acute lymphoid leukemias (ALLs), but not in other tumor types. In a subset of these mutations, functional analysis indicated defects in protein stability and localization. Taken together, these observations suggest that disruption of the ceramide pathway may contribute to a subset of human leukemias.

Description: Abstract 4176 We recently identified two members of the Groucho family of co-repressors, TLE (transducin-like enhancer of split) 1 and TLE4, as candidate tumor suppressor genes from the commonly deleted del(9q) region in acute myeloid leukemia (AML). This deletion is frequently associated with presence of the t(8;21) fusion gene AML1-ETO (RUNX1-RUNX1T1) and we have previously shown loss of these TLEs cooperates with AML1-ETO to increase survival, block differentiation, and induce myeloid cell proliferation. We have also shown that the proliferation and differentiation of several myeloid cell lines is markedly affected by modulation of TLE levels. Over-expression of TLEs induced THP-1 or HL-60 myeloid cell differentiation and blocks proliferation, while knockdown of TLEs triggers cell proliferation. We previously showed that knockdown of TLE1 and TLE4 inhibited all-trans retinoic acid (ATRA) induced differentiation of HL-60 cells. In this study we show knockdown of the TLEs also inhibits 12-O-tetradecanoyl-phorbol-13-acetate (TPA) induced differentiation of THP-1 cells, further pointing to the importance of these proteins in myeloid differentiation. Myeloid differentiation induced by TPA in THP-1 cells was associated with a transient translocation of TLE1 and TLE4 into the nucleus. We show that lowest levels of TLE1 and TLE4 are seen in the hematopoietic stem cell containing compartment of cord blood cells. We found a peak in TLE1/4 expression in early myeloid cells with lower levels in mature granulocytes. When we simultaneously knocked down both TLE1 and TLE4 we saw an expansion of the cord blood primitive progenitor population as measured by an increase of CD34+CD38- cells, 2.9 fold increase of colony forming units and 3.5 fold increase of Long-Term Culture-Initiating Cells. This data indicates low levels of TLE expression may maintain or expand undifferentiated hematopoietic progenitor cells and that a transient increase in their expression with translocation into the nucleus may trigger myeloid differentiation. We sought to further evaluate the mechanisms by which the TLEs regulate myeloid differentiation and proliferation. Using ImageStream analysis we found that TLE expression is able to inhibit activation of NFκB as evidenced by inhibition of nuclear NFκB translocation induced by lipopolysaccharide. Expression of the TLEs also reduces both of the nuclear translocation of β-catenin and total β-catenin in THP-1 cells. TLE1 inhibits LiCl-induced activation of Wnt signaling as measured by TOPFLASH. Knockdown of TLE1 and/or TLE4 in Kasumi1 cells induced expression of plakoglobin, a mediator of Wnt signaling. Blocking ATRA-induced myeloid differentiation by knocking down both TLE1 and TLE4 is associated with down-regulation of Nab2 and C/EBPe. In addition to the deletion of TLE1 and TLE4 in a subset of AMLs, epigenetic inactivation of TLE1 has also been reported in several myeloid malignancies, including AML. NF-κB and Wnt/β-catenin pathways in particular appear to be constitutively activated in AML and are believed to contribute to hematopoietic stem cell (HSC) expansion and increased survival. The loss of TLEs may contribute to the constitutive activation of these pathways in AML. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 1050 Poster Board I-72 Background: T-APL occurs frequently as a result of prior treatment with topoisomerase-II inhibitors (Mistry AR et al., NEJM, 2005). We have recently reported that the combination of all-trans retinoic acid (ATRA), ATO, with the addition of gemtuzumab ozogamicin for patients with a high presenting WBC, is effective in producing molecular remissions that are durable in APL (Ravandi F et al., JCO, 2009). Given the history of prior exposure to anthracycline chemotherapy in many patients with t-APL, an effective non-chemotherapy containing frontline regimen would be attractive in this patient population. Aim To examine the outcome of patients with t-APL, treated with ATO as part of the frontline regimen, and compare it to that of patients treated with standard ATRA plus anthracycline-based chemotherapy regimens. Method: We searched the database of the Leukemia Department at the University of Texas - M D Anderson Cancer Center (from 1980-2008) to identify patients with t-APL and define their characteristics and outcome. Results: Thirty six patients with t-APL were identified. Their median age was 54 years (range 26 to 81 years), and the median WBC count at presentation was 1,600/ul (range 500-162,500/ul). Most common prior malignancies were breast cancer (31%), prostate cancer (17%) and lymphomas (11%). Prior therapy included chemotherapy alone, radiation alone, or a combination of the two in 19%, 33%, and 47% of patients, respectively. Fifty percent of patients had been previously exposed to topoisomerase-II inhibitors. Median time from primary cancer to the diagnosis of t-APL was 3.5 years. The incidence of t-APL has increased with advancing decades: 9% of all patients with APL from 1980-99 vs. 16% from 2000-08 (P=0.07). Cytogenetic abnormalities in addition to t(15;17) occurred in 14/36 patients (39%) and most frequently involved chromosome 8 (4/36 patients, 11%). Among 25 patients with available PCR data, detection of the short PML-RARA isoform (14/25, 56%) was associated with a shorter survival compared to the long isoform (11/25, 44%) (161 weeks vs. 344 weeks; P= 0.29).The combination of ATO and ATRA (n=17) for induction resulted in a higher complete remission (CR) rate compared to ATRA plus chemotherapy (n=12) (88% vs. 67%; P= 0.35). The median overall survival for the patients treated with ATRA plus ATO was not reached compared to that for patients treated with ATRA plus chemotherapy (161 weeks; P=0.79). The median age for patients treated with ATRA plus ATO and ATRA plus chemotherapy was 53 and 56.5 years, respectively. The proportion of patients with a presenting WBC 〉 10,000/ul was 41% (7/17) in patients treated with ATRA plus ATO vs. 25% (3/12) in patients treated with ATRA plus chemotherapy. Conclusion: In our cohort of t-APL patients, outcomes with ATO and ATRA appear to be comparable to anthracycline containing induction regimens. This combination may be preferable in these patients in order to avoid any risk of anthracyclin-induced cardiomyopathy. Disclosures: Ravandi: Cephalon: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Description: Primary drug resistance is a major problem in multiple myeloma, an incurable disease of the bone marrow. Cell adhesion-mediated drug resistance (CAM-DR) causes strong primary resistance. By coculturing multiple myeloma cells with bone marrow stromal cells (BMSCs), we observed a CAM-DR of about 50% to melphalan, treosulfan, doxorubicin, dexamethasone, and bortezomib, which was not reversed by secreted soluble factors. Targeting the adhesion molecules lymphocyte function–associated antigen 1 (LFA-1) and very late antigen 4 (VLA-4) by monoclonal antibodies or by the LFA-1 inhibitor LFA703 reduced CAM-DR significantly. Only statins such as simvastatin and lovastatin, however, were able to completely restore chemosensitivity. All these effects were not mediated by deadhesion or reduced secretion of interleukin 6. Targeting geranylgeranyl transferase (GGTase) and Rho kinase by specific inhibitors (GGTI-298 and Y-27632), but not inhibition of farnesyl transferase (FTase) by FTI-277, showed similar reduction of CAM-DR. Addition of geranylgeranyl pyrophosphate (GG-PP), but not of farnesyl pyrophosphate (F-PP), was able to inhibit simvastatin-induced CAM-DR reversal. Our data suggest that the 3-hydroxy-3-methylglutaryl-coenzyme-A (HMG-CoA)/GG-PP/Rho/Rho-kinase pathway mediates CAM-DR and that targeting this pathway may improve the efficacy of antimyeloma therapies by reduction of CAM-DR.

Description: Deletions on chromosome 9q are seen in a subset of acute myeloid leukemia (AML) cases and are specifically associated with t(8;21) AML. We previously defined the commonly deleted region in del(9q) AML and characterized the genes in this interval. To determine the critical lost gene(s) that might cooperate with the AML1-ETO fusion gene produced by t(8;21), we developed a set of shRNAs directed against each gene in this region. Within this library, shRNAs to TLE1 and TLE4 were the only shRNAs capable of rescuing AML1-ETO expressing U937T-A/E cells from AML1-ETO–induced cell-cycle arrest and apoptosis. Knockdown of TLE1 or TLE4 levels increased the rate of cell division of the AML1-ETO–expressing Kasumi-1 cell line, whereas forced expression of either TLE1 or TLE4 caused apoptosis and cell death. Knockdown of Gro3, a TLE homolog in zebrafish, cooperated with AML1-ETO to cause an accumulation of noncirculating hematopoietic blast cells. Our data are consistent with a model in which haploinsufficiency of these TLEs overcomes the negative survival and antiproliferative effects of AML1-ETO on myeloid progenitors, allowing preleukemic stem cells to expand into AML. This study is the first to implicate the TLEs as potential tumor suppressor genes in myeloid leukemia.

Description: The Groucho (Gro)/TLE family of transcriptional co-repressor proteins have been called master regulatory genes based on their interaction with a variety of transcription factors and their critical role in development. The TLEs have also been shown to play major roles in brain and lymphocyte differentiation. We became interested in this gene family after finding two family members, TLE1 and TLE4, localized to the commonly deleted region on chromosome 9q in acute myeloid leukemia (AML). This deletion is tightly associated with t(8;21), and we recently showed loss of TLE1 and TLE4 cooperated with AML1-ETO to affect myeloid cell proliferation and survival, implicating TLE1 and 4 as potential tumor suppressor genes in AML. Based on their known ability to inhibit the function of several signaling pathways and transcription factors including Wnt/b-catenin, NF-kB, AML1, and Pu.1 known to be important in leukemogenesis and hematopoiesis, we undertook a series of experiments to determine whether the TLEs could affect myeloid cell differentiation and proliferation. We showed that expression of either TLE1 or TLE4 was able to induce differentiation in the HL-60 myeloid cell line as demonstrated by morphological changes, increased expression of the myeloid differentiation makers CD11b, CD14 and CD15, downregulated myeloperoxidase activity, as well as increased nitroblue tetrazolium (NBT) staining. Furthermore, when HL-60 cells were induced to differentiate by exposure to all-trans-retinoic acid (ATRA) we observed a 150-fold transient increase in TLE1 message after three days. Knockdown of TLE1 with specific shRNAs partially blocked ATRA-induced differentiation as monitored by CD11b expression, suggesting this burst of TLE1 expression is critical for ATRA induced myeloid cell differentiation. To determine the role of the TLEs in primary human cells, human cord blood cells were sorted, and TLE mRNA levels were determined in progenitor cells (CD34+/CD33−/CD16−/CD15−/CD14−), early myeloid precursors (CD34−/CD33+/CD16−/CD15−CD14−) and mature granulocytes (CD34−/CD33+/CD16+/CD15−CD14−). Lowest levels of TLE1 and TLE4 were found in progenitor cells with a peak of expression in early myeloid precursors. To help determine the significance of these low TLE levels in hematopoietic stem/progenitor cells, we designed shRNAs to simultaneously knockdown both TLE1 and TLE4. Knockdown of TLE1/4 in cord blood cells resulted in a 6.3-fold expansion of CD34+CD38− cells after 4 days’ coculture with M2-10B4 in the presence of hSCF, hFlt3-ligand, and hTPO, as compared with only 2.6-fold expansion of cells transfected with control shRNA. Similarly, using mouse bone marrow cells cultured in vitro in the presence of mIL-3, mIL-6 and mSCF, we found a 2.1-fold increase in mouse bone marrow Lin−/Sca-1+/c-kit+ (LSK) cells transfected with TLE1/4 shRNA as compared to cells transfected with scrambled control shRNA. Our results indicate that modulation of TLE levels is capable of influencing both myeloid differentiation, as well as expansion of the stem cell and/or progenitor population.

Description: We have recently developed a risk model specific for patients with lower risk MDS that allows the identification of poor prognosis patients (Leukemia. 2008; 22:538-3). We showed that the majority of patients classified by IPSS as low risk actually have poor outcomes with respect to overall survival. The cause of death (COD) of these patients is not well understood. In order to guide early therapeutic intervention, it is important to identify whether the mortality in this group is attributable to causes related to MDS or whether death occurs due to age-related comorbidities. The aim of the present study was to examine the rate of disease-related deaths in a cohort of 269 deceased patients with low or intermediate-1 disease by IPSS at presentation to a tertiary care center from 1980 to 2006. Autopsy, final progress note, physician letter, or death certificate were used to determine COD. For each patient, we categorized the COD as disease-related vs. non-related. MDS-related death was defined as infection, bleeding, transformation to acute myeloid leukemia (AML), or disease progression. Non-related causes included accidents, coronary events, heart failure, respiratory and renal failure, non-hemorrhagic stroke, and chronic hepatitis. Median age at presentation and at time of death was 66 years (range 19–88) and 67 years (range 21–90), respectively. Overall median survival was 59 weeks (range = 1–831). By FAB classification, all subgroups were identified (RA = 41.3%, RARS = 5.8%, RAEB = 28.6%, RAEB-t = 12.3%, CMML = 12.0%). By IPSS score, most patients were Intermediate-1 (Int-1 = 79% and Low = 21%). Most patients had diploid cytogenetics (58.3%), and the most common cytogenetic abnormality was deletion of chromosome 5 (9.1%). Almost all patients received supportive care only. The COD was identified as MDS-related in 229 of 269 patients (85%). The most common disease-related COD was infection (39% of all deaths) followed by hemorrhage (34%) and transformation to AML (23%). The most frequent non-disease-related COD were cardiovascular events (19 of 40 patients). 8 of 269 patients died of a secondary neoplasm. Our data demonstrates that the majority of patients with low- or intermediate risk MDS will succumb to causes related to their underlying disease. Although these results need to be validated prospectively, early therapeutic intervention could be considered in the management of these patients to improve survival.

Description: Deletion of the long arm of chromosome 9, del(9q), is one of the most common mutations associated with t(8;21) AML. Up to 50% of del(9q) AML is seen in association with t(8;21). The Runx1-ETO (AML1-ETO) fusion gene produced by t(8;21) is insufficient for leukemogenesis and loss of a critical gene(s) on chr9q apparently cooperates in leukemogenesis. We recently identified two members of the Groucho family of co-repressors, Transducin-like enhancer of split (TLE) 1 and TLE4, as candidate tumor suppressor genes, based on our mapping of the commonly deleted region in del(9q) AML. These proteins are known to inhibit Wnt signaling which has been implicated in hematopoietic stem cell renewal and they interact with hematopoietic transcription factors such as Runx1 and Pu.1. We have demonstrated that the expression of these two genes is specifically repressed in both del(9q) and t(8;21) AML samples. Knockdown of TLE1 or TLE4 mRNA using specific small interfering RNAs (siRNA) activates Wnt signaling in 293T cells as measured by TOPFLASH activity, while forced expression of TLE1 or 4 inhibits TOPFLASH. We show that siRNA against TLE4 using a specific siRNA increased cell cycle progression and cell division, while over-expression of TLE1 or TLE4 slowed cell cycle progression and lead to a pronounced growth disadvantage in THP-1 and HL60 myeloid cell lines. In addition to these effects on cell proliferation, these genes also affected myeloid cell differentiation. Over-expression of either TLE1 or TLE4 in monocytic THP-1 and promyelocytic HL-60 cell lines initiated myeloid differentiation as monitored by CD11b expression. These two genes have different effects on more terminal myeloid differentiation as TLE4, but not TLE1, was able to induce more terminal differentiation of THP-1 cells into monocytes as measured by CD14 expression, while TLE1 was able to induce the granulocytic marker CD15 in HL-60 cells. Furthermore, lentiviral delivery of siRNAs for either TLE1 or TLE4 to HL-60 cells inhibited induction of granulocytic and monocytic differentiation with all-trans retinoic acid (ATRA) or 1,25-dihydroxycholechalciferol (Vit.D3). TLE1 siRNA inhibited ATRA and Vit.D3 induced CD11b expression by more than 75%. While TLE4 siRNA decreased the induction of monocytic CD14 after Vit.D3 by 40%, TLE1 siRNA almost completely abrogated induction of CD14 with Vit.D3 or granulocytic CD15 with ATRA in HL-60 cells. Ex vivo culture of siRNA infected CD34 sorted human cord blood (CB) cells in semi-liquid media with differentiation inducing cytokines for seven days exhibited a decrease in the percentage of early CD33+ myeloid cells by 15% and 70% relative to controls in TLE1 and TLE4 siRNA infected CB cells, respectively. These results indicate that inhibition of TLE activity can promote cell cycle progression and inhibit myeloid differentiation. This study is the first to demonstrate a potential role for the TLEs in leukemogenesis and also indicates the TLEs may have non-redundant functions in myeloid differentiation. Understanding how various mutations work cooperatively to produce a malignant phenotype is one of the great challenges in oncology. The TLEs may represent an important cooperating mutation with Runx1-ETO in AML that links Wnt signaling and core binding protein transcription factors. Studies are currently underway to demonstrate this cooperativity.

Description: Abstract 2170 Background: ICH is a significant source of morbidity and mortality in pts with acute leukemias. Only relatively small retrospective series of pts with ICH have been reported previously. We evaluated the clinical characteristics of a large cohort of contemporary pts who developed ICH at MD Anderson Cancer Center (MDACC) to help better define the characteristics of pts at risk. Methods: We retrospectively searched the leukemia database at MDACC and identified 2421 pts who presented from 2005–2009 to MDACC with de novo acute myeloid leukemia (AML; n=1265), acute lymphoblastic leukemia (ALL; n=304), myelodysplastic syndrome (MDS; n=728), or blast-crisis CML (BC-CML; n=124). Clinical characteristics were obtained by chart review. Results: Among 2421 pts, a total of 119 pts developed ICH, as diagnosed by either CT or MRI brain (AML, n=72, 5.7%; ALL, n=19, 6.3%; MDS, n=13, 1.6%; CML-BC, n=15, 12.1%). Median age was 58years (range: 14–81), 58 (48.7%) were male, median WBC at the time of ICH was 2200/ul (0-186,000), platelet count 19,000/ul (0-318,000),hemoglobin 9.0 g/dl (4.5-14.7), peripheral blast count 0% (0-92%), INR 1.4 (0.4-12), PTT 29sec (17-66), creatinine 0.9mg/dl, and albumin 3.0g/dl (1.4-4.9). The localization of the bleed was intraparenchymal (IPH) in 58%, subdural (SDH) in 30%, subarachnoid (SAH) in 12%, and epidural (EDH) in 2.5%. Three pts had 2 different types of bleed. Median time from diagnosis to ICH was 210 days (0-1953). Only 18.5% of the patients bled within 30 days of diagnosis. Three patients had acute promyelocytic leukemia (APL), 2/3 bled within 2 days of diagnosis. Among 72 AML patients, the most common cytogenetic abnormality was del(5-, 7-) (26/72; 36.1%), and only 1/15 pts with diploid cytogenetics had a Flt3 point mutation. Surgical intervention for ICH was performed in 12 patients (10.1%). Median OS for all 119 patients was 40wks (0-257), and for those 12 undergoing surgery 42 wks (3-257). OS by type of bleed was as follows: IPH 33 wks (0-257), SDH 40 wks (1-174), SAH 41wks (1-86), EDH 8 wks(0-13). Conclusions: To our knowledge, we report here the largest cohort of contemporary pts with leukemia who develop ICH. While most previous studies highlight APL as the premier cause of ICH, in our cohort of over 2400 pts only 3 pts with APL developed ICH, likely due to heightened awareness for bleeding complications in these pts and aggressive transfusion. Pts in BC-CML have the highest relative proportion of ICH, suggesting a low threshold for head imaging in these pts. Most pts with ICH are profoundly thrombocytopenic at the time of the bleed, whereas the majority of them are not significantly coagulopathic. Less than 5% of all pts present with ICH at diagnosis or shortly thereafter, indicating that the major cause for developing ICH is chemotherapy-related thrombocytopenia or relapsed disease. Analyses to identify independent predictors of ICH in patients with leukemia are currently underway. The goal is to establish a novel predictive and/or prognostic model for ICH in patients with acute leukemias and MDS. Disclosures: Wierda: Abbott: Research Funding; Genentech: Honoraria, Speakers Bureau. Thomas: Novartis: Honoraria; Bristol-Meyer-Squibb: Honoraria; Pfizer:; Amgen: Research Funding. Kantarjian: AstraZeneca: Research Funding.

Description: Deletion of the long arm of chromosome 9, del(9q), is one of the most common cytogenetic abnormalities seen with t(8;21) AML. This close association suggests that that the loss of a critical gene(s) on chr9q cooperates with the AML1-ETO (RUNX1-MTG8) fusion gene produced by t(8;21) in leukemogenesis. We recently mapped the commonly deleted region of del(9q) AML to less than 2.4 Mb and identified all the genes mapping to this region. Extensive sequence analysis of these genes failed to reveal obvious mutations in del(9q) AML samples although the expression of a number of these genes appeared low specifically in del(9q) samples (Genes Chr and Ca 44, 279–291). To help determine the critical affected genes in the del(9q) CDR we used an siRNA expression library directed against all del(9q) CDR gene transcripts in an in vitro complementation assay with AML1-ETO. Inducible expression of AML1-ETO in U937 cells (U937T-A/E, Mol Cell Biol 21, 5577–5590) leads to cell cycle arrest and apoptosis. We introduced the del(9q) CDR siRNA library into U937T-A/E cells with the goal of finding siRNAs that rescued these cells after induction of AML1-ETO expression. The most commonly recovered siRNA using flanking PCR primers was directed against TLE1. Transducin-like enhancer of split (TLE) 1 and TLE4, are two members of the Groucho family of co-repressors that map adjacent to the del(9q) CDR. We subsequently demonstrated that knockdown of either TLE1 or TLE4 with specific siRNAs were able to overcome the induced cell cycle arrest in U937T-A/E cells. We further demonstrate that knockdown of either TLE in the AML1-ETO+ Kasumi-1 cell line increased cell division, increased expression of cyclin D1 and Ki67 and reduced the G0/G1 fraction, while expression of TLE1 or TLE4 in Kasumi-1 cells induced cell cycle arrest and apoptosis. Understanding how various mutations work cooperatively to produce a malignant phenotype is one of the great challenges in oncology. The TLEs are known to inhibit NFKB as well as Wnt signaling, two pathways implicated leukemic stem cell expansion. TLE inhibition may represent an important cooperating mutation with AML11-ETO in AML that links Wnt signaling and core binding protein transcription factors. Studies are currently underway to demonstrate this cooperativity and more directly evaluate the role of TLE inhibition in leukemogenesis.