ALBERT

Description: Introduction: p53 is an important tumor suppressor, and loss of p53 pathway function is a common event in human cancer. ASPP2 is a damage-inducible p53 binding protein that enhances apoptosis, at least in part, by selective stimulation of p53 pro-apoptotic target genes. Low ASPP2 expression occurs in many human tumors and as we have previously demonstrated, correlates with poor clinical outcome in patients with B-cell lymphomas. However, the mechanisms by which ASPP2 suppresses tumor formation remain unknown. Methods: To rigorously study ASPP2 in vivo function, we targeted the ASPP2 allele in a mouse model by homologous recombination by replacing exons 10–17 with a neoR gene. Aging mice were followed for spontaneous tumor formation. Additionally, six week old mice were irradiated at different doses and followed for tumor development. To explore the functional consequences of low ASPP2 expression, ASPP2+/− and ASPP2+/+ thymocytes were subjected ex vivo to 5Gy ionizing irradiation and apoptosis was assessed by Annexin-V/PI staining and flow cytometry. We also irradiated ASPP2+/+ and ASPP2+/−MEFs (mouse embryonic fibroblasts) with 5Gy and performed cell cycle analysis. Furthermore, we conducted global gene expression profiling between the unirradiated and irradiated ASPP2+/+ and ASPP2+/−MEFs using an Affymetrix GeneChip® Mouse Gene 1.0 ST Array. Moreover, phosphoproteomic analysis was performed on unirradiated and irradiated ASPP2+/+ and ASPP2+/−MEFs using 2-dimensional gel electrophoresis, fluorescent phosphoprotein dye Pro-Q Diamond staining, and liquid chromatography tandem mass spectroscopy. Results: We were unable to generate ASPP2−/− mice due to an early embryonic lethal defect. However, ASPP2+/− mice appear developmentally normal and reproduce. We observed an increased formation of spontaneous tumors in aging ASPP2+/− mice compared to ASPP2 +/+ mice (43% vs. 22%, at 115 weeks, p=0.011). Additionally we were able to show that after ionizing radiation, ASPP2+/− mice develop high-grade lymphomas in a dose-dependent manner at a significantly higher incidence at 50 weeks compared to ASPP2+/+ mice (p = 0.024 and p = 0.045, 6 Gy and 10.5 Gy respectively). Immunophenotyping demonstrated that these were high-grade T-cell lymphomas of thymic origin. Since ASPP2 is damage-inducible and promotes apoptosis, we wished to determine the extent to which reduction in ASPP2 expression attenuated the cellular damage response. We therefore gamma-irradiated ex vivo ASPP2+/+ and ASPP2+/−thymocytes in short-term culture and found a two-fold reduction in apoptosis in ASPP2+/− thymocytes compared to ASPP2+/+ thymocytes. Additionally, after 5 Gy gamma-irradiation, ASPP2+/− MEFs exhibited an attenuated G0/G1 checkpoint compared to ASPP2+/+ MEFs. Preliminary analysis of global gene expression in ASPP2+/+ and ASPP2+/− MEFs shows specific differences in gene expression patterns after damage. Likewise, preliminary analysis of phosphoproteomics between ASPP2+/+ and ASPP2+/− MEFs after damage, demonstrate differences in the phosphophorylation status of 170 proteins. Conclusions: ASPP2 is a haploinsufficient tumor suppressor, and reduction in ASPP2 expression attenuates both cell cycle checkpoints and apoptosis-induction after damage. These results suggest that reduction of ASPP2 levels modulate the cellular damage response, possibly at transcriptional as well as post-transcriptional levels, and provide a functional explanation for the increased tumor incidence in ASPP2+/− mice---since attenuated damage-response thresholds would lead to an impaired ability to eliminate cells that have accumulated tumorigenic mutations. Our study provides important insights into the p53-ASPP2 axis, and opens new avenues for investigation into its role in tumorigenesis and response to therapy.

Description: Fanconi anemia (FA) is an autosomal recessive disease characterized by congenital anomalies, aplastic anemia, and cancer susceptibility. Mutations within the FA complementation group C (FAC) gene account for approximately 14% of diagnosed FA cases. Two mutations, one in exon 1 (delG322) and one in exon 4 (IVS4 + 4 A to T), account for 90% of known FAC mutations. The delG322 mutation results in a mild FA phenotype, while the IVS4 + 4 A to T mutation results in severe FA phenotype. To determine the molecular basis for this clinical variability, we analyzed patient-derived cell lines for the expression of characteristic mutant FAC polypeptides. All cell lines with the delG322 mutation expressed a 50-kD FAC polypeptides, FRP-50 (FAC-related protein), shown to be an amino terminal truncated isoform of FAC reinitiated at methionine 55. All cell lines with the IVS4 + 4 A to T mutation lacked FRP-50. Overexpression of a cDNA encoding FRP-50 in an FA(C) cell line resulted in partial correction of mitomycin C sensitivity. In conclusion, expression of an amino terminal truncated FAC protein accounts, at least in part, for the clinical heterogeneity among FA(C) patients.

Description: Abstract 316 Background: The pathogenetic and high frequency D816V KIT mutation in aggressive systemic mastocytosis (ASM) and mast cell leukemia (MCL) exhibits in vitro and clinical resistance to imatinib. Midostaurin (PKC412) is an inhibitor of the KIT tyrosine kinase and can block D816V KIT-transformed cell growth at an IC50 of 30–40nM. Herein we report updated results of our fully accrued investigator-initiated, multicenter, phase II study of oral PKC412 in World Health Organization-defined ASM/MCL patients (pts). Method: PKC412 100 mg bid was administered as continuous 28-day cycles until progression/ intolerable toxicity. Lack of a major response (MR) or partial response (PR) per Valent criteria by the end of 2 cycles resulted in discontinuation from the protocol. Result: Efficacy and safety data for all 26 pts (15 male: 11 female) are presented. The distribution of SM pts with one or more “C” findings included ASM (n=4), SM-CMML (n=14), SM-MDS (n=3), SM-MDS/MPN-U (n=1), and MCL (n=4, two with associated MDS). The median age of pts was 62 years (range 24–79 years), and the median # of prior therapies was 1.5 (range 0–4). Responses have been observed in 18/26 pts (69%), consisting of 10 pts (38%) with a MR (6 incomplete remissions and 4 pure clinical responses), 5 pts (19%) with a good partial response (GPR), and 3 pts with a minor PR . Four pts had stable disease, and 4 pts exhibited progressive disease (PD). In subjects with higher quality responses, responses have been durable, with PKC412 administered for a median of 19.5 cycles (1.5 years) for MR pts and 15 cycles (1.2 years) for MR+PR pts (range 4+ - 58+ cycles). Major responses have included normalization of hypoalbuminemia, improvement of hemoglobin and platelet counts, and resolution of liver function abnormalities. High quality responses have also included near complete resolution of pleural effusion and ascites (with reduction of paracentesis), and substantial reversion or normalization of weight loss (including one pt discontinuing TPN). Clinically meaningful responses have been accompanied by reduction of palpable and/or 3-D volumetric CT measurement of hepato/splenomegaly, a 〉50% decrease in the serum tryptase level and/or marrow mast cell (MC) burden, and improvement in mediator symptoms and ECOG performance status. Cutaneous mast cell lesions faded in 2 pts. One patient with MCL has achieved a near complete remission with RBC transfusion independence, normalization of albumin, disappearance of 18 cm palpable splenomegaly (70% volume reduction by imaging), decrease of serum tryptase from 763 to 〈 20 ng/mL, and decrease of marrow MC burden from 60–70% to 5%. Of particular interest, in 3 pts with SM-CMML (all D816V KIT+), associated marked eosinophilia normalized (e.g. decreased from 50% to 1% in one pt).The most common grade 1–2 non-hematologic toxicities were nausea and/or vomiting, and less frequently diarrhea and fatigue. Asymptomatic and reversible hyperlipasemia occurred in 5 pts (grade 3, n=1). Hematologic toxicity with a suspected relationship to PKC412 has included ≥ grade 3 worsening of pre-existing anemia, and in one pt, recurrent grade 3 thrombocytopenia despite dose reduction to 50 mg bid. Dose reduction was undertaken in 4 additional pts (N/V, n=2; HA, n=1; transient increase in pre-existing pleural effusion, n=1). Dose re-escalation to 100 mg bid was feasible in 3 of these 4 pts. As of August 5, 2010, 8 pts continue PKC412, and treatment has been discontinued in 18 pts. Among the latter, 2 pts had PKC412 discontinued after 4 cycles (grade 3 fatigue, grade 2 N/V, n=1 each), and 7 initial responders have terminated therapy for PD after 8–39 cycles (1 for progression of CMML). Allele-specific PCR detected D816V KIT in 18/26 (69%) pts, and a novel, activating two–amino acid insertion in MPL was identified in a D816V KIT-negative pt. A statistically significant association was observed between positivity for D816V KIT and achieving a MR versus any other type of response (p=0.0095, Fisher's exact test). Pharmacokinetic data and histopathologic correlates of response (e.g. marrow MC burden and serum tryptase levels) will be presented. Conclusion: PKC412 is well tolerated and exhibits clinically relevant and durable responses which compare very favorably to literature reports of interferon-alpha or cladribine in advanced SM. An ongoing international trial aims to further evaluate the efficacy and safety of PKC412 in this pt population. Disclosures: Gotlib: Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding. George:Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding. Dutreix:Novartis: Employment. Gross:Novartis: Employment. Nikolova:Novartis: Employment. Graubert:Novartis: Research Funding.

Description: Stimulation of the erythropoietin receptor (EPO-R) or the interleukin-2 receptor (IL-2-R) by their respective ligands has been reported to activate tyrosine phosphorylation of the cytoplasmic protein, Shc. We have recently characterized a cell line, CTLL-EPO-R, that contains functional cell-surface receptors for both EPO and IL-2. Although stimulation with IL-2 or IL-15 resulted in the rapid, dose-dependent tyrosine phosphorylation of Shc, stimulation with EPO failed to activate Shc. EPO, IL-2, and IL-15 activated the tyrosine phosphorylation of the adaptor protein, Shp2, and the association of Shp2/Grb2/cytokine receptor complexes. In addition, EPO, IL-2, and IL-15 activated Raf1 and ERK2, demonstrating that the Raf1/MEK/MAP kinase pathway was activated. These results indicate that multiple biochemical pathways are capable of conferring a mitogenic signal in CTLL-EPO-R. EPO can activate the Raf1/MEK/ MAP kinase pathway via Shc-dependent or Shc-independent pathways, and Shc activation is not required for EPO-dependent cell growth in CTLL-EPO-R.

Description: Vascular endothelial growth factors (VEGF) and their receptors (VEGFR) deliver important signals that controlling the angiogenesis of growing, injured, and malignant tissues. Expression of VEGF receptors has been described on a variety of normal and malignant leukocytes, and presumably plays a role in their trafficking to injured tissue. Differential expression of neuropilin-1 (NRP-1) has been described as a marker of regulatory T cells (Treg) in mice, and may be expressed on human thymocytes, but expression on human Tregs has not been confirmed. Moreover, earlier reports of Treg-specific markers in humans were based on the CD4+CD25+ subset, which is not as reliable for Treg identification as it is in mice. We used cell sorting to obtain pure populations of human Tregs (using the more stringent criteria of CD4+CD25+CD125−) and T helper cells (Th) (CD4+CD25−CD127+). We compared expression in these subsets of VEGFR-1, -2, -3, and NRP-1 and -2 using flow cytometry and quantitative RT-PCR, and NRP-1 splice isoforms by RT-PCR using isoform-specific primers. We were not able to identify any NRP-1 expression in Tregs by any of these methods. In contrast, there was marked differential expression of VEGFR-2 (Flk-1) and -3 (Flt-4), both of which exhibited up to 16-fold higher expression in Tregs than in Th. These findings suggest a role for VEGF in recruiting Tregs to sites of tissue growth as a mechanism for blunting potential autoimmunity to neoexpressed antigens, and thus may also explain the accumulation of Tregs in tumors.

Description: Background: Constitutively activated receptor tyrosine kinase (TK) D816V KIT contributes to the pathogenesis of most ASM cases and exhibits resistance to imatinib. Midostaurine (MST, PKC412) is an inhibitor of the KIT TK and can block D816V KIT-transformed cell growth at an IC50 of 30–40nM. We report updated results of our ongoing phase II study of oral MST in ASM patients (pts). Methods: MS 100 mg bid was administered as continuous 28-day cycles until progression/intolerable toxicity. Lack of a major response (MR) or partial response (PR) per Valent criteria after 2 cycles resulted in pt discontinuation. To date, 15 pts have been enrolled, 9 with an associated MDS/MPD. Median age was 62 yrs (range 24–76), median # prior therapies was 1 (0–3). All 15 pts are evaluable for efficacy and safety. Responses were observed in 11/15 pts (73%). Best responses included 5 pts with a MR (all incomplete remissions), and 6 pts with a PR (5 good, 1 minor). Four pts were stopped after 2 cycles (2 stable disease, 2 progressive disease (PD)). The 5 MRs included resolution of hypoalbuminemia (n=3), increase in hemoglobin to 〉10 g/dL (n=1), and normalization of mild splenomegaly by CT (n=1). Additional MR findings in these pts included improvement in platelets to 〉100,000/mm3, and normalization of LFTs. Good PRs included reduction of ascites (n=2), 〉50% reduction in palpable hepatosplenomegaly (n=1), and 〉50% reduction in direct hyperbilirubinemia (n=2). In MR/PR pts, other findings included marked reduction in pleural effusion (n=2), reversion of weight loss, and improvement in cutaneous mastocytosis (MC) lesions. Among responders, the median duration of treatment was 10 cycles (4–18+). In 4 pts, the marrow MC burden decreased from the 50–60% to 10–15% range (stable in other responders). There was inconsistent reduction in the serum tryptase level among MR/PR pts. Safety: Nausea and/or vomiting (N/V) was the most frequent non-hematologic toxicity. Grade 1 tremor, grade 1–2 diarrhea, fatigue, and headache (HA) were less common. Possibly related hematologic toxicity consisted of worsening anemia in 2 pts (grade 3), and recurrent grade 3 thrombocytopenia despite dose reduction to 50 mg bid. Dose reduction occurred in 4 additional pts (N/V, n=2; HA, n=1; recurrent pleural effusion, n=1). Dose re-escalation to 100 mg bid was feasible in 3 of these 4 pts. Two pts had MST discontinued after 4 cycles (grade 3 fatigue, grade 2 N/V, n=1 each), and after cycles 8 and 15 for PD (n=2). Real-time allele-specific PCR has preliminarily detected D816V KIT in 9/15 (60%) pts. Further accrual is required to render any correlation between KIT mutation status and response. Preliminary pharmacokinetic analysis reveals that plasma concentrations of MST reached a maximal level of ∼3462 ng/mL on day 3, gradually decreased with time, and reached a stable level of ∼1368 ng/mL after cycle 1. This time dependent pattern is consistent with previous findings in AML pts. In conclusion, Midostaurine has sustained partial remitting activity in a high proportion of ASM pts.

Description: Accumulating evidence indicates that bone marrow is a source of endothelial progenitor cells. Recently our laboratory has shown that functional endothelial cells (EC) can be clonally derived from phenotypically defined hematopoietic stem cells. To determine the endothelial cell potential of human hematopoietic cells, blood vessels in skin and gut biopsies from sex-mismatched allogeneic transplant recipients were examined. H&E sections were initially reviewed to ensure tissue integrity and the absence of significant graft versus host disease. Coded biopsy specimens from 12 sex-mismatched transplant recipients and 6 sex-matched female transplant recipients were evaluated in a blinded fashion. Endothelial cells were identified using a combination of immunohistochemistry (IHC) and XY interphase FISH. Tissues sections were first evaluated by IHC for EC markers (CD31 or vWF) and for the pan-hematopoietic marker CD45 then assayed by dual-color interphase FISH for donor derived cells (XY). Tissue samples from each recipient were assayed at least 3 times by sequential IHC/FISH. The frequency of CD31+, CD45−, Y+ cells and vWF+, CD45−, Y+ cells in biopsies from the 18 transplant recipients was evaluated by 2 independent observers. 9 of 12 sex-mismatched recipients showed evidence of donor-derived Y chromosome positive EC. The remaining 3 recipients did not exhibit hematopoietic engraftment at the time of biopsy. Donor derived endothelial cells were detected in the skin and the gut of transplant recipients with a mean frequency of 2.3% (range 0.9–3.9). Donor derived hematopoietic cells including intraepithelial lymphocytes and tissue macrophages were excluded by CD45 expression. None of the 〉4,000 endothelial cells examined had 〉2 sex chromosomes, consistent with an absence or very low incidence of cell fusion. In addition, Y chromosome signals were not detected in sex matched female recipients, thereby excluding vertical transmission of male cells. Furthermore, none of the recipients evaluated prior to hematopoietic engraftment had detectable donor derived endothelial cells, indicating a close linkage between the recovery of hematopoiesis and endothelial cell outcomes. Our results suggest that the close association between hematopoiesis and the vascular system originally identified during early development persists into adult life. Consequently it will be important to study the potential therapeutic role of bone marrow derived endothelial cells in the setting of hematopoietic and vascular disease.

Description: Stimulation of the erythropoietin receptor (EPO-R) or the interleukin-2 receptor (IL-2-R) by their respective ligands has been reported to activate tyrosine phosphorylation of the cytoplasmic protein, Shc. We have recently characterized a cell line, CTLL-EPO-R, that contains functional cell-surface receptors for both EPO and IL-2. Although stimulation with IL-2 or IL-15 resulted in the rapid, dose-dependent tyrosine phosphorylation of Shc, stimulation with EPO failed to activate Shc. EPO, IL-2, and IL-15 activated the tyrosine phosphorylation of the adaptor protein, Shp2, and the association of Shp2/Grb2/cytokine receptor complexes. In addition, EPO, IL-2, and IL-15 activated Raf1 and ERK2, demonstrating that the Raf1/MEK/MAP kinase pathway was activated. These results indicate that multiple biochemical pathways are capable of conferring a mitogenic signal in CTLL-EPO-R. EPO can activate the Raf1/MEK/ MAP kinase pathway via Shc-dependent or Shc-independent pathways, and Shc activation is not required for EPO-dependent cell growth in CTLL-EPO-R.

Description: Apoptosis Stimulating Protein of p53 (ASPP2) binds the tumor suppressor protein p53 and promotes damage-induced apoptosis in part through stimulation of p53 apoptotic function. We have previously demonstrated that low ASPP2 levels correlate with poor clinical outcome in patients with diffuse large B-cell lymphoma treated with anthracycline-based chemotherapy, which is consistent with its role as a tumor suppressor. We targeted the ASPP2 allele in a mouse model by homologous recombination using a knockout vector that replaced exons 10–17 with a neoR gene. Although ASPP2−/− is an early embryonic lethal event, ASPP2+/– mice appeared developmentally normal and reproduce. Importantly, ASPP2+/– mice developed tumors spontaneously at a higher frequency than sibling ASPP2+/+ controls. We now show that after ionizing irradiation, ASPP2+/– mice develop high grade lymphatic malignancies compared to ASPP2+/+ mice. ASPP2+/+ and +/– mice were irradiated, with either 6 Gy or 10,5 Gy, 8 weeks after birth. Following irradiation, mice were monitored weekly by blood smears and flow cytometry if suspicious cells were observed. Tumor formation was detected as early as 17 weeks post-irradiation. In the 10,5 Gy group, ASPP2+/– mice developed thymic lymphomas (7/31), while no tumors were detected in ASPP2+/+ mice (0/14). Tumor-free survival was statistically significant (p= 0,045, log-rank test). In the 6 Gy dose group 9 out of 28 of the ASPP2+/– mice developed high-grade thymic lymphomas compared to 0/20 ASPP2+/+ mice. Tumor-free survival was statistically significant (p=0,024, log-rank test). Immunophenotyping and flow cytometry on mice with circulating tumor cells from whole unfractionated gated viable bone marrow (Bm) cells revealed that the Bm was typically almost completely replaced by CD4neg, CD5+, CD8+ leukemic/lymphoma cells that are negative for B-cell (B220) and myeloid markers (Mac-1/Gr-1). Preliminary analysis demonstrated that MEFs (mouse embryonic fibroblasts) from ASPP2+/– mice treated with doxorubicin, serum starvation or irradiation had an attenuated G1/G0 checkpoint arrest. Ongoing experiments are being conducted in thymocytes prepared from mice of different ASPP2 and p53 genotypes. Our data demonstrates that ASPP2 plays an important role in suppressing the development of lymphomas after irradiation in a mouse model. Consequently, disruption of the p53-ASPP2 axis may be important in lymphomagenesis and response to therapy. Furthermore, our data provides a rationale for the future study of ASPP2 expression in patients during the formation of secondary hematologic malignancies after therapies with DNA damaging agents.

Description: Background: Resistance to imatinib in patients with chronic myeloid leukemia (CML) is commonly associated with reactivation of BCR-ABL due to kinase domain mutations or increased expression. Activation of SRC family kinases has been shown in isolated patients without evidence of BCR-ABL reactivation. We hypothesized that activating mutations of the RAS pathway (one of the major downstream mediators of BCR-ABL) or of leukemia-associated tyrosine kinases other than BCR-ABL may cause BCR-ABL-independent resistance. Patients and Methods: CML patients with imatinib resistance were initially screened for BCR-ABL mutations. Two sets of nested primers were used to specifically amplify BCR-ABL. Sequence analysis covered the entire 5′ end of ABL including the Cap, SH3, SH2 and kinase domains. Bone marrow samples from patients without BCR-ABL mutations (N=17) were screened for mutations of NRAS, KRAS and FLT3 by direct DNA sequencing. Analysis of PTPN11 and PDGFR is ongoing and will be presented. Results: We detected a KRAS exon 3 mutation (T58I) in 1/17 patients (5.8%) and confirmed this mutation by 2-directional sequencing of 3 independently amplified PCR products. No mutations in NRAS or FLT3 were observed. Analysis of archived samples revealed that T58I was absent while the patient was responding to 800mg of imatinib and achieved a major cytogenetic response (MCyR), became detectable before relapse and remained detectable (at approximately 30%) in 4 subsequent samples collected over a period of 18 months (Table 1), while the patient achieved a second MCyR on dasatinib. The T58I allele became undetectable prior to relapse on dasatinib. None of the samples tested were positive for BCR-ABL kinase mutations. T58I is an activating mutation of KRAS that has been detected in patients with Noonan syndrome who developed JMML (Schubbert et. al, Nature Genetics, 2006, 38:331). We tested the functional relevance of T58I in the context of imatinib resistance in 32D cells co-expressing T58I and BCR-ABL, with KRAS G12D and native KRAS as controls. Sensitivity to imatinib was reduced (G12D〉T58I〉native), with 15% 32D cells expressing KRAS T58I retaining viability at 2.5 μM over 3 Days. These data demonstrate that T58I confers partial imatinib resistance, although it does not induce complete growth factor independence. Conclusion: Activating KRAS mutations may contribute to resistance or disease persistence in CML patients treated ABL kinase inhibitors. Table 1: Follow-up summary of patient with KRAS T58I mutation Sample date Treatment prior to cytogenetic evaluation (mg/day) Cytogenetics (Ph+ metaphases) FISH (Bcr-Abl)% KRAS T58I Allele% Bcr-Abl kinase domain Feb-02 Imatinib 400 11/17 41.5 0 WT Dec-02 Imatinib 400 20/20 78 0 WT Dec-03 Imatinib 600–800 +Arsenic trioxide 5/25 31.5 0 WT Oct-04 Imatinib 800+Arsenic trioxide 7/20 14.5 31.8 WT Jun-05 Imatinib 800 17/20 62.5 ND WT Oct-05 Dasatinib 140 ND 23 28.8 WT Dec-05 Dasatinib 100 6/20 24.5 ND WT Mar-06 Dasatinib 80 1/1 13.5 37.4 WT Apr-06 Dasatinib 80 1/12 10 ND WT Sep-06 Dasatinib 80 3/20 24 34.9 WT Dec-06 Dasatinib 80 1/3 29 33.1 WT Feb-07 Dasatinib 80 4/20 51 0 WT May-07 Dasatinib 80 19/20 60.5 0 WT