Silencing HDAC3 Enhances Apoptosis of Acute B Lymphoblastic Leukemia Cells smurf2 Inducing By Inhibiting JAK/STAT3 Signal Pathway

Abstract Purpose Smad ubiquitin regulatory factor 2(Smurf2) as a member of ubiquitinated E3 ligase was certificated to regulate the ubiquitination level of target gene. In this study, smurf2 as tumor suppressor was studied in acute B lymphoblastic leukemia, Histone deacetylases 3(HDAC3) which was connected with smurf2 was related with poor prognosis. It is further proved that smurf2 and HDAC3 were expected to be therapeutic targets for B-ALL cells and improve the prognosis of patients with B-ALL. Methods Realtime-PCR and westernblot were used to detect the expression of smurf2 and HDAC3 in healthy donors and B-ALL patients by us. Sup-B15 and CCRF-SB were treated with MG-132, small interfering RNA of smurf2 or HDAC3. Up-regulating smurf2 plasmid was also transfected to B-ALL cells by lipo3000. Flow cytometry and westernblot were used to validate the difference of apoptosis and cell cycle. Results Smurf2 mRNA in B-ALL patients was significantly lower than that in healthy donors. HDAC3 was higher in B-ALL patients than that in healthy donors. Protein level of smurf2 and HDAC3 was consistent with mRNA level. Up-regulating smurf2 by plasmid or MG-132 and silencing smurf2 could influence HDAC3, which further depressed JAK/STAT3 signal pathway inducing apoptosis of B-ALL cells. Sup-B15 and CCRF-SB were treated with siHDAC3 and MG-132 for 24h, we discovered that silencing HDAC3 enhanced the apoptosis level of B-ALL cells MG-132 inducing by depressing JAK/STAT3 pathway. It was expected to become a therapeutic target for improving the clinical prognosis of B-ALL patients. On the other hand, we found that MG-132 caused G2/M phase arrest in B-ALL cells and successfully inhibited the JAK/STAT3 pathway leading to apoptosis. Conclusions Silencing HDAC3 inhibited the JAK/STAT3 pathway and further enhanced apoptosis in B-ALL cells MG-132 inducing. HDAC3 and smurf2 were expected to become therapeutic targets for clinical treatment of acute B lymphocytic leukemia and improve survival rate of leukemia patients. Key words Smurf2; HDAC3; Apoptosis; Prognosis; Disclosures No relevant conflicts of interest to declare.

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Akt Inhibitors Induce Apoptosis in Chronic Lymphocytic Leukemia Cells.

Blood ◽

10.1182/blood.v114.22.1731.1731 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1731-1731

Author(s):

Mercè de Frias ◽

Daniel Iglesias-Serret ◽

Ana M Cosialls ◽

Llorenç Coll-Mulet ◽

Antonio F Santidrián ◽

...

Keyword(s):

T Cells ◽

Chronic Lymphocytic Leukemia ◽

Conflicts Of Interest ◽

Therapeutic Option ◽

Mrna Level ◽

Lymphocytic Leukemia ◽

Mrna Levels ◽

Dependent Manner ◽

Healthy Donors ◽

Induced Apoptosis

Abstract Abstract 1731 Poster Board I-757 Phosphatidylinositol-3-kinase (PI3K)/Akt pathway has been described to be critical in the survival of chronic lymphocytic leukemia (CLL) cells. Here, we have analyzed the effect of two selective chemical inhibitors of Akt (Akti-1/2 and A-443654) in the survival of CLL cells. We studied by cytometric analysis the cytotoxic effects of Akt inhibitors on peripheral B and T lymphocytes from patients with CLL and from healthy donors. Both inhibitors induced apoptosis in CLL cells in a dose-dependent manner. Moreover, B cells from CLL samples were more sensitive to Akt inhibitors than T cells from CLL samples, and B or T cells from healthy donors. Survival factors for CLL cells, such as IL-4 and SDF-1a, were not able to block the apoptosis induced by both Akt inhibitors. We studied the changes induced by Akti-1/2 and A-443654 at mRNA level by performing reverse transcriptase multiplex ligation–dependent probe amplification (RT-MLPA). Akti-1/2 did not induce any change in the mRNA expression profile of genes involved in apoptosis, while A-443654 induced some changes, including an increase in NOXA and PUMA mRNA levels, suggesting the existence of additional targets for A-443654. We also studied the changes induced by both Akt inhibitors in some BCL-2 protein family members on CLL cells by Western blot. Both inhibitors induced an increase in PUMA and NOXA protein levels, and a decrease in MCL-1 protein level. Moreover, Akti-1/2 and A-443654 induced apoptosis irrespective of TP53 status. These results demonstrate that Akt inhibitors induce apoptosis of CLL cells and might be a new therapeutic option for the treatment of CLL. Disclosures No relevant conflicts of interest to declare.

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Anlotinib Shows Potent Antileukemia Effects in B-Cell Acute Lymphocytic Leukemia through the Blockage of Angiogenic Related Pathways

Blood ◽

10.1182/blood-2020-141039 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 49-49

Author(s):

Qiuling Chen ◽

Yuelong Jiang ◽

Qinwei Chen ◽

Long Liu ◽

Bing Xu

Keyword(s):

B Cell ◽

Solid Tumors ◽

Molecular Mechanisms ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Unmet Need ◽

Lymphocytic Leukemia ◽

Advanced Lung Cancer ◽

Antitumor Effects

Acute lymphoblastic leukemia (ALL) derives from the malignant transformation of lymphoid progenitor cells with ~85% being originated from B-cell progenitors (B-ALL). Despite fairly good prognoses for most pediatric B-ALL patients, the outcome is fatal in over 50% of adult patients who have a recurrent or progressive disease and lack of effective therapeutic approaches. Therefore, novel treatment strategies with high efficacy and low toxicity are an unmet need for B-ALL patients, especially those with relapsed or refractory status. Angiogenesis is a process of new vessel formation that requires the participation of multiple proangiogenic factors (e.g., VEGF, PDGF, and FGF) and their corresponding receptors (e.g., VEGFR, PDGFR, and FGFR). Angiogenesis, a well-established feature of solid tumors, also contributes to leukemia progression and correlates with the involvement of specific sanctuary sites in ALL, highlighting that the perturbation of angiogenesis would be an attractive approach for ALL treatment. Anlotinib is an oral tyrosine kinase (TKI) inhibitor with a broad range of antitumor effects via the suppression of VEGFR, PDGFR and FGFR. Of importance, anlotinib has been approved for the treatment of advanced lung cancer in China. Here, we evaluated the antileukemia activity of anlotinib in preclinical B-ALL models and its underlying molecular mechanisms. In this study, we observed that anlotinib significantly blunted the capability of cell proliferation and arrested cell cycle at G2 phase in B-ALL cell lines. Subsequently, we found that anlotinib resulted in remarkably enhanced apoptosis in B-ALL in vitro. To assess the in vivo antileukemia potential, we established a B-ALL patient-derived xenograft (PDX) mouse model and then treated the B-ALL PDX model with anlotinib. As a result, oral administration of anlotinib pronouncedly delayed in vivo B-ALL cell growth and reduced leukemia burden with acceptable safety profiles in this model. As for the mechanism of action, the antileukemia effect of anlotinib was associated with the disruption of the role of VEGFR2, PDGFRb, and FGFR3. Moreover, we revealed that this drug blocked the PI3K/AKT/mTOR/ signaling, a pathway that is linked with angiogenesis and its proangiogenic regulators, including VEGFR2, PDGFRb, and FGFR3. In aggregate, these results indicate that anlotinib is a potent antitumor agent for the treatment of B-ALL via the inhibition of angiogenic relevant pathways, which provide a novel potential treatment intervention for patients with B-ALL who have little effective therapy options. Disclosures No relevant conflicts of interest to declare. OffLabel Disclosure: Anlotinib originally designed by China is a novel orally active multitarget inhibitor that is evaluating in clinical trials against multiple solid tumors.

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Use of Ubiquitin-Proteasome System Profiling for Differentiating Between Various Leukemic Processes.

Blood ◽

10.1182/blood.v114.22.4712.4712 ◽

2009 ◽

Vol 114 (22) ◽

pp. 4712-4712

Author(s):

Ke Zhang ◽

Hagop M. Kantarjian ◽

Wanlong Ma ◽

XI Zhang ◽

Xiuqiang Wang ◽

...

Keyword(s):

Myeloid Leukemia ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Blast Crisis ◽

Chronic Phase ◽

Direct Analysis ◽

Cell Types ◽

Ubiquitin Proteasome System ◽

Lymphocytic Leukemia ◽

Ubiquitin Proteasome

Abstract Abstract 4712 The ubiquitin-proteasome system (UPS) plays a major role in cell homeostasis in normal and neoplastic states. Expression and function of the UPS system vary with the specific characteristics of individual cell types, suggesting that determination of UPS “signatures” could be useful in identifying various cell populations. Since direct analysis of cancer cells is often problematic, even in hematologic diseases, we explored the potential of using UPS signatures in plasma to differentiate between various leukemias. We first analyzed plasma UPS profiles of patients with acute myeloid leukemia (AML; n=111), acute lymphoblastic leukemia (ALL; n=29), advanced myelodysplastic syndrome (MDS; n=20), chronic lymphocytic leukemia (CLL; n=118), or chronic myeloid leukemia (CML; n=128; 46 in accelerated/blast crisis [ACC/BL], 82 in chronic phase), and 85 healthy control subjects. Plasma levels of proteasome, ubiquitin (poly-ubiquitin), and the 3 proteasome enzymatic activities (chymotrypsin-like [Ch-L], caspase-like [Cas-L], trypsin-like [Tr-L]) were measured. Specific activities were calculated by normalizing each of the 3 enzyme activities to the levels of proteasome protein in plasma (Ch-L/p, Cas-L/p, and Tr-L/p). These 8 variables were used in multivariate logistic regression models to differentiate between leukemic processes. UPS signatures provided clear differentiation between patients with a leukemic process and normal controls (AUC=0.991), using 6 different variables (Tr-L/P, Ch-L, Ch-L/p, Cas-L, Cas-L/P, ubiquitin). Distinguishing between acute (AML, ALL, MDS) and chronic (CML, CLL) processes was less efficient (AUC=0.853 using Tr-L, Tr-L/P, Cas-L/P, Ch-L/P, proteasome, Ch-L), likely due to the high proportion (36%) of CML patients in ACC/BL phase. However, UPS signatures generally yielded powerful differentiation between individual leukemias (Table). MDS was not well differentiated from AML (AUC=0.791), reflecting the significant biological overlap of these diseases. These data support the potential usefulness of the UPS profile to aid in the differential diagnosis of various leukemias. Disclosures: No relevant conflicts of interest to declare.

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Quantitative Expression Analysis of WT1 Main Isoforms in AML

Blood ◽

10.1182/blood.v118.21.1469.1469 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1469-1469

Author(s):

Irene Luna ◽

Esperanza Such ◽

Jose Cervera ◽

Eva Barragan ◽

Marta Llop ◽

...

Keyword(s):

Cord Blood ◽

Peripheral Blood ◽

Conflicts Of Interest ◽

Wilms Tumor ◽

Mrna Level ◽

Prognostic Impact ◽

Cell Selection ◽

Healthy Donors ◽

Cd34 Cells ◽

Cord Blood Cd34

Abstract Abstract 1469 The Wilms Tumor 1 (WT1) gene was first described as a tumour suppressor gene, but its accurate role in leukemia development has not been completely elucidated. Some authors support the role of WT1 as a prognostic marker in acute myeloid leukemia (AML) based on the assessment of its expression at the mRNA level. However, the prognostic value of the main isoforms of WT1 has been less studied. The aim of this study was to develop a specific quantitative assay to estimate the ratio of expression of the four major WT1 isoforms (A, 5-/KTS-; B, 5+/KTS-; C, 5-/KTS+; D, 5+/KTS+) and to evaluate their prognostic impact. WT1 expression was analyzed in bone marrow samples from 108 patients with AML at diagnosis (65 male/46 female, median age: 61 yr, range: 17 – 91). Likewise, peripheral blood samples of 20 healthy donors and 6 samples of cord blood CD34+ cell selection were analyzed as normal controls. We performed a new method to quantify the ratios of the four major isoforms of WT1. Briefly, to amplify all isoforms within a PCR reaction, specific WT1 primers flanking exon 4 to exon 10 were used in cDNA samples, followed by capillary electrophoresis with laser-induced fluorescence analysis on an ABIPRISM 310 DNA Analyzer (Applied Biosystems, Foster City, CA) and lastly analyzed with the Gene Mapper 4.2 software (Applied Biosystems). The amount of each isoform was calculated by the area under the curve. Subsequent comparisons of isoform ratios were made by standardized calculation of percentage. All values are given as the mean of duplicate PCRs. In parallel, RQ-PCR for total WT1 detection was performed as previously described by Barragan et al. (Haematologica 2004; 89: 926–933). GUS gene was used as housekeeping gene. Eighteen patients (17%) did not express WT1, while 90 patients (83%) overexpressed WT1 above background levels. The median value of each WT1 isoform was: 18% (range: 2 – 73) for A isoform; 16% (range: 7 – 63) for B isoform; 24% (range: 2 – 52) for C isoform; and 33% (range: 3 – 55) for D isoform. None of healthy donors had detectable WT1 levels in peripheral blood. All samples of CD34+ cells expressed the four isoforms of WT1: 21% (range: 2 – 26) for A isoform; 16% (range: 1 – 64) for B isoform; 24% (range: 1 – 47) for C isoform; and 36% (range: 25 – 44) for D isoform. These data reveal that, in our series, the most predominant isoform was +5/+KTS, both in AML and in cord blood CD34+ cell selection samples. There were no significant differences when comparing the proportion of each isoform between the cord blood CD34+ cell selection samples and the cohort of AML patients. There was not significant correlation between the overexpression of total WT1 with the ratio of each isoform, and we were unable to demonstrate that the overexpression of WT1 is due to a particular isoform overexpression. A significant lower event-free survival (EFS) was observed in those patients overexpressing total WT1, taking a cut-off value of 3000 WT1 copies/ GUS copies × 104 (75th percentile, P =.001). However, when the same cut-off as well as the median value for each one of the isoforms was used, we found no significant differences in EFS and in overall survival. To sum up, none of the isoforms were correlated with overexpression of total WT1 or survival. We were unable to find differences between the expression of each isoform of WT1 in CD34+ cells from normal cord blood and in AML patients. Further studies including larger controls need to be carried out. Disclosures: No relevant conflicts of interest to declare.

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SALL4 Is a Key Survival Factor in Acute B-Cell Lymphoblastic Leukemia

Blood ◽

10.1182/blood.v118.21.2428.2428 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2428-2428

Author(s):

Shikiko Ueno ◽

Jiayun Lu ◽

He Jie ◽

Ailing Li ◽

XiaoXian Zhang ◽

...

Keyword(s):

B Cell ◽

Cell Lines ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Mrna Level ◽

Down Regulation ◽

Immunodeficient Mice ◽

Donor Cells ◽

Reh Cells ◽

And Control

Abstract Abstract 2428 SALL4 is a zinc-finger transcriptional factor and a member of the SALL gene family. It plays an essential role in the maintenance of ESC pluripotent and self-renewal properties by interacting with other two key regulators in ESCs, Nanog and Oct4. We previously have shown that stem cell factor SALL4 is aberrantly expressed in 75% of acute B-cell lymphoblastic leukemia (B-ALL). We have also shown that SALL4 is aberrantly expressed in AML, and down-regulation of SALL4 in AML leads to significant cell death. In this study, we focused on investigating the functional role of SALL4 in human B-ALL leukemogenesis. We first assessed the SALL4 mRNA level in four B-ALL cell lines (REH, Nalm6, 697, Blin-1) and five primary patient samples by qRT-PCR. We observed SALL4 mRNA in these four cell lines increased when compared to normal human CD34 negative BM cells. Moreover 4 of 5 primary samples showed high level expression of SALL4, suggesting that SALL4 might play a role in B-ALL pathogenesis. Then, we selected a SALL4 expressing B-ALL cell line (REH and Nalm6) and attenuated SALL4 expression through GFP-labeled shRNA approach in these cell lines. We monitored the growth of SALL4 knockdown and control REH and Nalm6 cells through MTS assay. SALL4 knockdown cells had a decreased growth rate compared to that of the control cells. We also stained SALL4 knockdown and control cells with Annexin V and 7-AAD by flow cytometric quantitation of apoptotic cells. The percentages of apopotic cells in SALL4 knockdown cells were much higher than these in controls. These data demonstrated that inhibition of SALL4 in REH cells and Nalm6 cells led to reduced proliferation and increased apoptosis. We then examined the oncogenesis ability of SALL4 knockdown REH cells in a mouse xenotransplantation model. SALL4 knockdown or control REH cells were injected intravenously into immunodeficient mice. All the recipients succumbed to fatal leukemia within 4 to 6 weeks post transplantation. In both BM and spleen of SALL4 knockdown recipients the engrafted proportion of GFP+ cells was significantly decreased compared to the initial donor cells. Whereas, in both BM and spleen control recipients the percentage of GFP+ REH cells engrafted was similar to that of initial donor cells. This suggests that down-regulation of SALL4 is essential for B-ALL engraftment. To rule out the observed engraftment defect was due to homing, we next performed homing assay. SALL4 knockdown or control cells were injected intravenously into immunodeficient mice as well. Three hours of the injection, mice were sacrificed and analyzed the percentage of GFP+ cells in BM and spleen by flow cytometry. There was no difference among SALL4 knockdown and the control. Furthermore, we performed gene expression profiling on apoptosis-related genes in SALL4 knockdown and control REH cells. The result showed that in SALL4-knockdown REH, TNF mediated cell apoptosis pathways was up-regulated as well as multiple caspase members. The expression of Caspase 3, Caspase 8, FADD was up-regulated in both SALL4 knockdown REH and Nalm6 when compared to these controls, and was verified by real time RT-PCR. This suggests that SALL4 could repress apoptosis through the TNF signal pathway. In summary, we report a novel SALL4/TNF pathway in maintaining cell survival in B-ALL. Disclosures: No relevant conflicts of interest to declare.

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Expression of PIM-2 and NF-κB Is Increased in Patients with Acute Myeloid Leukemia (AML) and Acute Lymphoblastic Leukemia (ALL) and Correlates with Complete Remission Rate

Blood ◽

10.1182/blood.v118.21.4648.4648 ◽

2011 ◽

Vol 118 (21) ◽

pp. 4648-4648

Author(s):

Katarzyna Kapelko-Slowik ◽

Donata Urbaniak-Kujda ◽

Dariusz Wolowiec ◽

Jaroslaw Dybko ◽

Bozena Jazwiec ◽

...

Keyword(s):

Complete Remission ◽

Conflicts Of Interest ◽

Remission Rate ◽

Lymphoblastic Leukemia ◽

Pcr Analysis ◽

Serine Threonine Kinase ◽

Primary Resistance ◽

Signalling Molecules ◽

Healthy Donors ◽

Acute Myeloid

Abstract Abstract 4648 Background. PIM-2 is a proto-oncogene that encodes for serine/threonine kinase which interacts with various signalling molecules. PIM-2 is highly expressed in neoplastic tissues and in leukaemic and lymphoma cell lines which is consistent with a its role during oncogenic transformation. The nuclear factor kappa B (NF-κB) pathway appears to be deregulated in variety of tumors, with sustained activity of NF-κB leading to apoptotic resistance in tumor cells The aim. The aim of this study was to investigate whether the PIM-2 and NF-κB expression is altered in acute myeloid leukaemia (AML) and acute lymphoblastic leukaemia (ALL). Patients and methods. One hundred fourty-three patients were included: 91 with AML and 52 with ALL (42 with B-ALL and 10 with T-ALL), aged 18–84 (median=41). Seventy-five patients reached complete remission (CR): 50 in AML and 25 in ALL. Bone marrow samples were collected at the time of diagnosis. Control samples were obtained from 24 healthy donors. We analysed PIM-2 and NF-κB expression by RQ-PCR analysis. Results. Expression of both PIM-2 and NF-κB in all leukaemic patients and in subgroups: AML and ALL was significantly higher than in controls. In AML group patients who reached CR expressed PIM-2 and NF-κB at significantly lower levels than patients with primary resistance to chemotherapy (with no CR, NCR). Moreover in AML, we have found the correlation between PIM-2, NF-κB expression and blasts in myelogram and PIM-2 and patients‘ age. Summary. Our data indicate that PIM-2 and NF-κB genes expression is increased in patients with AML and ALL and correlates with CR rate in AML patients. Disclosures: No relevant conflicts of interest to declare.

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Acute Lymphocytic Leukemia and Down Syndrome

Blood ◽

10.1182/blood.v118.21.sci-8.sci-8 ◽

2011 ◽

Vol 118 (21) ◽

pp. SCI-8-SCI-8

Author(s):

Shai Izraeli

Keyword(s):

Down Syndrome ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Mtor Inhibitors ◽

Lymphocytic Leukemia ◽

Leukemic Cells ◽

Aberrant Expression ◽

Childhood All ◽

Increased Risk ◽

The Common

Abstract SCI-8 Children with Down syndrome are at a markedly increased risk for acute lymphoblastic leukemia (DS-ALL). These leukemias are exclusively of the B lymphoid precursor phenotype and occur in a similar age to “common” sporadic ALLs with the striking absence of infant ALL. Recent studies reveal that DS-ALLs are heterogeneous and differ from sporadic ALLs. Only about a fifth of DS-ALLs carry the common cytogenetic aberrations typical to childhood ALL. Genomic rearrangements leading to the expression of a cytokine receptor, CRLF2, are detected in 60% of DS-ALL in comparison with up to 10% of sporadic ALLs. CRLF2 heterodimerizes with Interleukin 7 receptor-α (IL7R) to form the receptor to thymic stromal lymphopoietin (TSLP). This receptor is usually present in macrophages, dendritic cells, and some T lymphocytes and participates in allergic and inflammatory processes. The aberrant expression of the TSLP receptor is DS-ALL (and sporadic ALL) is often associated with additional mutations that cause constitutive activation of the downstream JAK-STAT and mTOR growth signaling pathways. These are either lymphoid specific activating mutations of JAK2 or JAK1 or mutations in CRLF2 or IL7R that cause ligand-independent receptor dimerization. The role of the trisomy in selecting these somatic abnormalities is presently unknown. Clinically, the prognosis of DS-ALL is inferior to sporadic ALL mainly because of increased treatment toxicity. However, recent data suggest that the inferior outcome may also be related to the genetic properties of the leukemic cells and that excessive chemotherapy dose reduction may not be appropriate for these patients. Therefore increased vigilance for infectious complications and optimal supportive care are required during periods of intensive chemotherapy. The common activation of the TSLP signaling pathway in DS-ALLs suggests a future for targeted therapy with JAK and/or mTOR inhibitors. Importantly, research of DS-ALL has proven relevant for the general patient population with ALL, as somatic mutations in the TSLP pathway have been discovered in children and adults with sporadic ALL. A major research challenge is the elucidation of the roles of constitutional and somatic trisomy 21 in leukemogenesis. Disclosures: No relevant conflicts of interest to declare.

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SF3B1 Mutations in CLL Are Equivalent to p53/ATM Dysfunction and Cause Defective Puma Upregulation in Response to Chemotherapy

Blood ◽

10.1182/blood.v120.21.711.711 ◽

2012 ◽

Vol 120 (21) ◽

pp. 711-711 ◽

Cited By ~ 2

Author(s):

Doreen te Raa ◽

Ingrid AM Derks ◽

Dieuwertje M Luijks ◽

Jacoline van Laar ◽

Hanneke Monsuur ◽

...

Keyword(s):

Poor Prognosis ◽

Target Genes ◽

Conflicts Of Interest ◽

Lymphocytic Leukemia ◽

Splicing Factor ◽

Clinical Prognosis ◽

Mrna Induction ◽

Response To Chemotherapy ◽

Recurrent Mutations

Abstract Abstract 711 Mutations or deletions of the tumor suppressor p53 or its upstream kinase ATM are well-known determinants of poor prognosis in Chronic Lymphocytic Leukemia (CLL). In recent years, genome wide sequencing has uncovered novel gene mutations that correspond with poor prognosis. Specifically, recurrent mutations in the splicing factor SF3B1 and the Notch and NRAS/KRAS oncogenes have been found. These mutations were (in part) mutually exclusive with p53 and/or ATM mutations, which suggested overlap in biological function. Here, we report results of a comparative analysis of p53 target genes and in vitro drug responses in CLL samples with either p53 (n=9), ATM (n=10), SF3B1 (n=11), Notch (n=6), or NRAS/KRAS (n=4) gene deletions/mutations. We found that upon irradiation, mRNA induction of all tested p53 targets genes (p21, Puma, CD95, Bax, PCNA, FXDR) was clearly decreased in all SF3B1 mutated CLL samples (overall p<0.001). SF3B1 samples resembled ATM mutated/11q− CLL in displaying a defective but not absent p53 response. In contrast, Notch and KRAS/NRAS mutations did not affect RNA induction of apoptosis inducers Puma and Bax. At the protein level, Puma and p21 induction were defective or absent in SF3B1 mutated CLL. This corresponded with decreased apoptosis after in vitro treatment with fludarabine. Treatment with nutlin, either alone or in combination with fludarabine, restored cell death induction, again indicating an overlap with ATM dysfunction. To establish possible causality between SF3B1 mutation and ATM dysfunction, more genetic and functional studies are ongoing and will be reported. In conclusion, the recently described mutations in a splicing factor in CLL can be linked at the functional level to defective ATM and/or p53 target gene responses, providing an explanation for the poor clinical prognosis of CLL patients with SF3B1 mutations. Disclosures: No relevant conflicts of interest to declare.

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Comparison Of Mutational Profiles Of Diagnosis and Relapsed Pediatric B-Acute Lymphoblastic Leukemia: A Report From The COG ALL Target Project

Blood ◽

10.1182/blood.v122.21.824.824 ◽

2013 ◽

Vol 122 (21) ◽

pp. 824-824 ◽

Cited By ~ 3

Author(s):

Jinghui Zhang ◽

Mignon L. Loh ◽

Xiaotu Ma ◽

Michael Rusch ◽

Gang Wu ◽

...

Keyword(s):

Acute Lymphoblastic Leukemia ◽

Exome Sequencing ◽

Somatic Mutations ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Therapeutic Targets ◽

Purine Metabolism ◽

White Blood Count ◽

Ras Signaling ◽

Sequencing Data

Abstract Characterization of the genetic landscape of relapsed pediatric acute lymphoblastic leukemia (ALL) and changes that occur with disease progression provides insight into the molecular basis of relapse and may identify new therapeutic targets. We analyzed 20 diagnosis-remission (germline)-relapse trio samples of pediatric B-ALL by high-coverage (>200x) whole-exome sequencing. Included patients were originally NCI high risk (HR) by either age (≥10 years) or white blood count (≥50,000/microliter), enrolled on a Children’s Oncology Group B-ALL trial, and experienced bone marrow relapse. Samples were selected based upon availability of sufficient high quality material from all three time points. We identified recurrent relapse-specific somatic mutations in 5 genes with significant frequency found in genes encoding the purine 5’ nucleotidase NT5C2 (n=7, 35%) and the histone acetyltransferase CREBBP (n=2, 10%). Furthermore, we discovered novel recurrent somatic mutations that were highly enriched in relapsed ALL (20%) compared with diagnosis (5%) in WHSC1, USH2A and NT5C1B, another enzyme involved in purine metabolism. Three of the four WHSC1 mutations cause the same amino acid change E1099K in the highly conserved SET domain in which structural modeling predicts perturbed WHSC1-substrate interactions resulting in increased WHSC1 activity. The WHSC1 and NT5C2 mutations are mutually exclusive with a combined prevalence of 55% in relapsed tumors. Analysis of a validation cohort of 63 independent trios from both NCI standard risk and HR cohorts replicated the findings in NT5C2 (n=8, 13%) and WHSC1 E1099K (n=6, 10%). Five pathways were significantly mutated at relapse with high-frequency somatic mutations present at diagnosis and/or relapse of the 20 ALL trio samples: the Ras signaling pathway (NRAS, KRAS, PTPN11, FLT3; 65%), genes involved in histone modification (MLL2, WHSC1, SETD2, CREBBP; 50%), purine metabolism (NT5C2 and NT5C1B; 45%), tyrosine kinase signaling (JAK2, CRLF2; 25%) and genes regulating B-cell development (PAX5, IKZF1; 15%). The median number of non-silent coding region sequence mutations in diagnostic samples was 10 (range 4-25) while that of the relapse samples was 25 (range 7-506) including 3 hypermutated samples with >100 non-silent mutations accompanied by a dominance of C(G)>T(A) substitution in relapse-specific mutations, suggesting a possibility that these mutations may be induced by a specific mutagen. Most of the diagnostic and relapse tumors were polyclonal based on diagnosis-relapse comparison of mutant allele fraction (MAF). Inter-tumor MAF of a recurrently mutated gene was highly heterogeneous despite an estimated >70% leukemia involvement for most specimens, suggesting presence of subclonal mutations. For example, NT5C2 MAF in the relapsed specimens ranges from a low of 0.08 to a high of 0.93. Ten relapsed specimens demonstrated evolution from a minor subclone (<10%) in the diagnostic specimen; 8 of the subclones have oncogenic mutations in NRAS, KRAS, JAK2, WHSC1 or CRLF2. A notable finding was the lack of preservation of specific clonal RAS pathway mutations from diagnosis to relapse as subclonal mutations in KRAS, PTPN11 and FLT3 present in diagnosis were replaced by a dominant NRAS mutation in relapse. We were also able to identify structural alterations present in both relapse and diagnosis tumors from exome sequencing data, including IGH@-CRLF2 fusion (n=2), BTG1 deletion (n=2), ETV6-RUNX1 fusion (n=1), intragenic deletion of RUNX1 (n=1) and MAP3K2 (n=1), focal amplification in the last exon of MYC (n=1) and a t(1;14)(p36;q32) translocation resulting in truncation of SLC2A5 and BTBD7. These results provide new insights into the genetic events contributing to the relapse of pediatric B-ALL, and suggest new potential therapeutic targets. Disclosures: No relevant conflicts of interest to declare.

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Risks and Benefits of Sex-Mismatched Hematopoietic Cell Transplantation Differ By Conditioning Intensity

Blood ◽

10.1182/blood.v124.21.2537.2537 ◽

2014 ◽

Vol 124 (21) ◽

pp. 2537-2537

Author(s):

Hideki Nakasone ◽

Mats Remberger ◽

Lu Tian ◽

Petter Brodin ◽

Bita Sahaf ◽

...

Keyword(s):

Multivariate Analyses ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Chronic Gvhd ◽

Lymphocytic Leukemia ◽

Hematopoietic Cell ◽

University Hospital ◽

Cell Transplant ◽

Increased Risk ◽

Conditioning Intensity

Abstract Backgrounds: Sex-mismatched hematopoietic cell transplant (HCT), especially HCT of male recipients with female donors (F->M) is known to be associated with a higher incidence of graft-versus-host disease (GVHD) and inferior survival. Total lymphoid irradiation with anti-thymocyte globulin (TLI-ATG) has been reported to reduce GVHD incidences and non-relapse mortality (NRM). We hypothesized that TLI-ATG could reduce adverse effects of sex-mismatched HCT without reducing graft-versus leukemia/lymphoma (GVL) effect. Methods: We reviewed clinical charts of 1041 adult recipients who received peripheral blood stem cell transplant between 2006 and 2013 at Stanford University (n=749) and Karolinska University Hospital (n=292). Primary diseases included acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), lymphoma, chronic lymphocytic leukemia (CLL) and others. Our study excluded indications for which TLI-ATG is not used such as acute lymphoblastic leukemia. Haplo-identical HCT and recipients who received GVHD prophylaxis other than cyclosporine and tacrolimus were also excluded. Impacts of sex-mismatch on clinical outcomes were separately assessed in TLI-ATG (n=437), reduced-intensity conditioning (RIC, n=266), and myeloablative conditioning (MAC, n=345). We also measured allo-antibodies (HY-Ab) against 5 HY-antigens encoded on the Y-chromosome (DBY, UTY, ZFY, EIF1AY, and RPS4Y) at 3 months (3m) post F->M HCT in the Stanford cohort. Results: F->M HCT was significantly associated with an increased risk of acute GVHD grade 2-4 in the RIC group (HR 1.96, P<0.01) and an increased risk of chronic GVHD in the MAC group (HR 1.83, P<0.01), while no impact of sex-mismatch on GVHD was observed in the TLI-ATG group. F->M HCT was also associated with an increased risk of NRM only in the MAC group (HR 1.84, P=0.022), while there was no difference in the TLI-ATG or RIC group. In the TLI-ATG group, relapse incidences of sex-mismatched HCT at 2 years post-HCT (40% in F->M HCT and 33 % in M->F HCT) were significantly lower than that of sex-matched HCT (52%, P<0.01, Fig1). Multivariate analyses revealed that sex-mismatch was significantly associated with reduced relapse in the TLI-ATG group (HR 0.64 in F->M and 0.59 in M->F, P<0.01 in each), while no difference was observed in the RIC or MAC group. The GVL benefit of sex-mismatch in TLI-ATG was observed in AML and lymphoma patients other than CLL. In MDS and CLL patients, the GVL benefit by sex-mismatch was not observed. We found that the overall survival (OS) of sex-mismatch recipients (69% in F->M and 70% in M->F HCT at 2 years post-HCT) was higher than that of sex-matched HCT (56%, P<0.01, Fig 1) in the TLI-ATG group. In contrast, for MAC recipients, OS of F->M HCT (49%) was significantly inferior to those of sex-matched HCT (60%) and M->F HCT (58%, P=0.01). Multivariate analyses confirmed that sex-mismatch was significantly associated with superior OS in the TLI-ATG group (HR 0.69 in F->M, P=0.037; HR 0.61 in M->F, P=0.014), while F->M HCT was significantly associated with inferior OS in the MAC group (HR 1.59, P=0.018). In the TLI-ATG group, the benefit of sex-mismatched HCT on OS seems due to the reduced relapse rate. We previously reported HY-Ab response post-HCT was associated with chronic GVHD as a representative of allo-Ab response (Nakasone et al. ASH 2013). We then hypothesized that HY-Ab response 3m post-HCT could predict reduced relapse in F->M HCT with TLI-ATG. Excluding patients with MDS and CLL because of the absence of GVL benefit by sex-mismatch in TLI-ATG (above), relapse incidence at 2 years post-HCT was higher in the recipients who had no HY-Ab response at 3m post-HCT vs. those who did (49% vs. 26%, P=0.037, Fig 2). Multivariate analysis corroberated that the detection of HY-Ab 3m post-HCT was significantly associated with reduced relapse in F->M HCT with TLI-ATG (HR 0.29, P=0.039). On the other hand, in the MAC group, HY-Ab 3m post-HCT was not significantly associated with reduced relapse. Conclusion: Benefits and risks of sex-mismatch differ according to conditioning intensity. Recipients of TLI-ATG conditioning preferentially benefit from sex-mismatched HCT with significantly reduced relapse rates and improved OS. HY-Ab 3m post-HCT as a representative of allo-Ab demonstrated the association with reduced relapse in the TLI-ATG group. We believe that sex-mismatched HCT should be selected in TLI-ATG, while F->M HCT should be avoided in MAC. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures No relevant conflicts of interest to declare.

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