Loss Of p53 Induces Leukemic Transformation In a Murine Model Of JAK2V617F-Induced Polycythemia Vera

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 269-269
Author(s):  
Takako Tsuruta-Kishino ◽  
Keisuke Kataoka ◽  
Hiroshi Kobayashi ◽  
Junji Koya ◽  
Kensuke Narukawa ◽  
...  
Keyword(s):  
Polycythemia Vera ◽  
Research Funding ◽  
Myeloproliferative Neoplasms ◽  
Myeloid Cells ◽  
Pulmonary Hemorrhage ◽  
Leukemic Cells ◽  
Erythroid Progenitors ◽  
Leukemic Transformation ◽  
Erythroid Precursors

Abstract Myeloproliferative neoplasms (MPN) have an inherent tendency toward leukemic transformation, but its mechanisms remain largely unknown. Recently, TP53 mutation is reported to be frequently found in cases with post-MPN leukemia. Here, to address the contribution of p53 loss to leukemic transformation from MPN in vivo, we retrovirally transduced c-kit+ bone marrow (BM) cells from p53 knockout (p53-/-) and littermate mice (p53+/+) with either wild-type Jak2 (Jak2WT) or Jak2V617F respectively, and transplanted them into lethally irradiated mice. At 3 weeks after transplantation, both recipients of Jak2V617F/p53-/- and Jak2V617F/p53+/+ cells developed a polycythemia vera-like disease characterized by high WBC count and elevated hemoglobin (Hb) level. Jak2V617F/p53+/+ mice survived and continued to have elevated Hb level, whereas 5 weeks after transplantation, Jak2V617F/p53-/- recipients developed cachexia, and their Hb level declined. Eventually, these mice developed fatal leukemia with a median survival of 46.5 days after transplantation, suggesting loss of p53 cooperates with Jak2V617F mutation to promote leukemic transformation from MPN. To characterize these leukemias, we analyzed leukemic tissues from moribund Jak2V617F/p53-/- mice. Peripheral blood smears and BM specimen from Jak2V617F/p53-/- recipients showed a marked increase of erythroid precursors with dysplastic features, leading to suppression of normal hematopoiesis. Notably, Jak2V617F/p53-/- mice displayed marked hepatosplenomegaly and extensive pulmonary hemorrhage. Consistent with the histopathologic findings, Jak2V617F/p53-/- animals exhibited a remarkable accumulation of erythroid precursors (CD71+), and especially more immature progenitors (Ter119-/CD71+) in the BM and spleen, compared with Jak2V617F/p53+/+ animals. These data suggest Jak2V617F/p53-/- recipients developed infiltrative disease with accumulation of immature erythroid cells, fulfilling the Bethesda Criteria of erythroleukemia in mice. To assess the transplantability of Jak2V617F/p53-/- leukemia, we injected unfractionated BM cells from Jak2V617F/p53-/- mice into lethally irradiated mice. In all cases, lethal leukemia developed earlier than in primary recipients. Moreover, there was a significant increase in erythroid progenitors in secondary recipients, suggesting the erythroid component is the predominant lineage involved in this leukemia model. As Jak2V617F/p53-/- leukemic tissues contained three major populations: CD71+ erythroid progenitors, Mac1+ mature myeloid cells, and lineage-negative (CD71-/Mac1-) primitive leukemic cells, we purified and transplanted these subfractions into secondary recipients to evaluate their leukemia-initiating potential. As a result, both lineage-negative (CD71-/Mac1-) cells and CD71+ erythroid progenitors possessed leukemia- initiating capacity, but Mac1+ myeloid cells could not reconstitute the disease. In addition, these two fractions had different capacities to induce leukemias; recipients of CD71+ cells rapidly developed erythroleukemia, whereas lineage-negative cells caused lethal leukemia after the polycythemic state. Moreover, hematopoietic tissues in recipients transplanted with CD71+ cells mainly consisted of erythroid lineages, whereas lineage-negative cells produced both erythroid and myeloid lineages, suggesting lineage-negative cells are more immature than CD71+ erythroid precursors. Furthermore, subsequent fractionation of lineage-negative cells revealed leukemia-initiating cells were enriched in Lin-/Sca-1+/c-kit+ (LSK) cells. To further characterize two types of leukemia-initiating cells in Jak2V617F/p53-/- leukemia, we assessed their sensitivity to a JAK2 inhibitor, INCB18424, in vitro. Interestingly, INCB18424 treatment significantly reduced CD71+ cell proliferation, whereas LSK cells were able to expand in the presence of INCB18424, indicating different leukemia-initiating cells existing in post-MPN leukemia have different responsiveness to JAK2 inhibiton. In summary, these results demonstrate p53 loss is sufficient for inducing leukemic transformation in JAK2V617F-postive MPN and offers an in vivo model to assess novel therapeutic approaches for post-MPN leukemia. In addition, we revealed leukemia-initiating cells at different differentiation stages could exist in post-MPN leukemia. Disclosures: Kurokawa: Novartis: Consultancy, Research Funding; Bristol-Myers Squibb: Research Funding; Celgene: Consultancy, Research Funding.

Development of a Murine Model for Leukemic Transformation of Myeloproliferative Neoplasms for Preclinical Therapeutic Studies

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 808-808 ◽  
Author(s):  
Raajit K. Rampal ◽  
Suveg Pandey ◽  
Omar Abdel-Wahab ◽  
Jennifer J Tsai ◽  
Taghi Manshouri ◽  
...  
Keyword(s):  
Murine Model ◽  
Research Funding ◽  
Somatic Mutations ◽  
Myeloproliferative Neoplasms ◽  
Survival Curve ◽  
Philadelphia Chromosome ◽  
In Vivo Studies ◽  
Leukemic Transformation

Abstract Abstract 808 A subset of patients with Philadelphia-chromosome negative myeloproliferative neoplasms (MPNs) (Polycythemia Vera (PV), Essential Thrombocytosis (ET), and Primary Myelofibrosis (PMF)) subsequently transform to acute myeloid leukemia (AML). Leukemic transformation (LT) after MPN occurs in as many as 23% of PMF patients within 10 years of diagnosis, and in 4–8% of PV and ET patients in the first 18 years after diagnosis. The development of AML after an antecedent MPN is associated with a dismal clinical outcome, and is associated with a poor response to conventional anti-leukemic therapies. Although somatic mutations in the JAK-STAT signaling pathway, including in JAK2 and MPL, occur in the majority of MPN patients, the somatic mutations that drive LT from a pre-existing MPN have not been fully delineated. Recent candidate mutational studies have identified recurrent somatic mutations in a subset of known leukemogenic disease alleles at the time of transformation from MPN to AML, including mutations in TP53, IDH1/2, TET2 and SRSF2 as well as deletions in IKZF1. However, the functional contribution of these specific genetic events to LT has not been delineated, and genetically accurate models of transformation of Philadelphia-chromosome negative MPN to AML have not been reported to date. In order to develop a genetically accurate murine model of LT, we have modeled expression of JAK2V617F mutation in combination with TP53 loss in vivo to further our understanding of progression from MPN to AML and to use this preclinical model of LT to test novel therapies. Bone marrow (BM) cells from C57/Bl6 Tp53−/− and littermate control mice were infected with JAK2V617F-IRES-GFP retrovirus, followed by transplantation of transduced cells into lethally irradiated congenic recipients. Of note, transplantation of JAK2V617F/Tp53−/− cells, but not JAK2V617F positive cells was associated with impaired survival; 50% of mice injected with JAK2V617F/Tp53−/− cells died by day 100, whereas all mice injected with JAK2V617F positive cells survived 100 days or longer (p=0.011) (figure 1). Mice injected with JAK2V617F/Tp53−/− cells presented with significant leukocytosis, with a mean WBC of 38.4 in mice engrafted with JAK2V617F/Tp53−/− cells compared with 11.4 in JAK2V617F/Tp53 wildtype mice. At the time of sacrifice, all mice engrafted with JAK2V617F/Tp53−/− cells had increased numbers of blasts in the peripheral blood and bone marrow, as assessed by morphologic evaluation and flow cytometric analysis which noted CD117 expression on leukemic blasts. BM cells from mice engrafted with JAK2V617F/Tp53−/− cells were characterized by increased serial replating (>10 platings), which was not observed in plating studies with JAK2V617F positive cells. In addition, we noted that the disease from JAK2V617F/Tp53−/− cells, but not JAK2V617F positive cells, was transplantable into secondary recipients consistent with increased self-renewal in vivo. We have begun testing the efficacy of novel therapies in this murine model, using both in vitro assays and in vivo studies in secondary transplantation studies. Treatment with the JAK kinase inhibitors INCB18424 and CYT 387 resulted in dose-dependent inhibition of colony formation in vitro. The combination of INCB18424 and Decitabine (which has demonstrated clinical efficacy in post-MPN-AML) is associated with synergistic inhibitory effects in vitro. Based on these results, we are performing in vivo studies with INCB18424, Decitabine, and INCB18424 + Decitabine, and results from these preclinical therapeutic studies will be presented in detail. Taken together, our data demonstrate that expression of JAK2V617F plus Tp53 loss, a genoptype commonly seen in patients who transform to AML after MPN, efficiently models LT in vivo. This model can now be utilized to examine the mechanisms of leukemic transformation, including assessment of the leukemic cell of origin in transformed disease. In addition this model can be utilized to test novel therapeutic strategies in a preclinical setting, which can be used to inform clinical trials in this poor-risk hematologic malignancy. Figure 1: Survival curve of mice transplanted with JAK2V617F in presence and absence of Tp53 Figure 1:. Survival curve of mice transplanted with JAK2V617F in presence and absence of Tp53 Disclosures: Verstovsek: Incyte Corporation: Research Funding; Novartis: Research Funding; AstraZeneca: Research Funding; Celgene: Research Funding; SBIO: Research Funding; Lilly Oncology: Research Funding; Bristol-Myers: Research Funding; Geron Corp.: Research Funding; Gilead: Research Funding; YM Biosciences: Research Funding; Roche: Research Funding; NS Pharma: Research Funding; Infinity Pharmaceuticals: Research Funding.

In Vitro Expansion of Polycythemia Vera Progenitors Favors Expansion of Erythroid Precursors without JAK2 V617F Mutation.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3506-3506 ◽  
Author(s):  
Josef T. Prchal ◽  
Ko-Tung Chang ◽  
Jaroslav Jelinek ◽  
Yongli Guan ◽  
Amos Gaikwad ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Polycythemia Vera ◽  
Peripheral Blood ◽  
Jak2 V617f ◽  
Jak2 V617f Mutation ◽  
Erythroid Progenitors ◽  
Erythroid Precursors ◽  
In Vitro Expansion ◽  
V617f Mutation

Abstract A single acquired point mutation of JAK2 1849G>T (V617F), a tyrosine kinase with a key role in signal transduction from growth factor receptors, is found in 70%–97% of patients with polycythemia vera (PV). In the studies of tyrosine kinase inhibitors on JAK2 1849G>T (see Gaikwad et all abstract at this meeting) we decided to study the possible therapeutic effect of these agents using native in vitro expanded cells from peripheral blood. To our surprise, the in vitro expansion of PV progenitors preferentially augmented cells without JAK2 1849G>T mutation. We used a 3 step procedure to amplify erythroid precursors in different stages of differentiation from the peripheral blood of 5 PV patients previously found to be homozygous or heterozygous for the JAK2 1849G>T mutation. In the first step (days 1–7), 106/ml MNCs were cultured in the presence of Flt-3 (50 ng/ml), Tpo (100 ng/ml), and SCF (100 ng/ml). In the second step (days 8–14), the cells obtained on day 7 were re-suspended at 106/ml in the same medium with SCF (50 ng/ml), IGF-1 (50 ng/ml), and 3 units/ml Epo. In the third step, the cells collected on day 14 were re-suspended at 106/ml and cultured for two more days in the presence of the same cytokine mixture as in the step 2 but without SCF. The cultures were incubated at 37oC in 5% CO2/95% air atmosphere and the medium renewed every three days to ensure good cell proliferation. The expanded cells were stained with phycoerythrin-conjugated anti-CD235A (glycophorin) and fluorescein isothiocyanate-conjugated anti-human-CD71 (transferrin receptor) monoclonal antibodies and analyzed by flow cytometry. The cells were divided by their differential expression of these antigens into 5 subgroups ranging from primitive erythroid progenitors (BFU-Es and CFU-Es) to polychromatophilic and orthochromatophilic erythroblasts; over 70% of harvested cells were early and late basophilic erythroblasts. The proportion of JAK2 1849G>T mutation in clonal PV granulocytes (GNC) before in vitro expansion and in expanded erythroid precursors was quantitated by pyrosequencing (Jelinek, Blood in press) and is depicted in the Table. These data indicate that in vitro expansion of PV progenitors favors expansion of erythroid precursors without JAK2 V617F mutation. Since three PV samples were from females with clonal granulocytes, erythrocytes, and platelets, experiments were underway to determine if the in vitro expanded erythroid cells were clonal PV cells without JAK2 V617F mutation, or derived from polyclonal rare circulating normal hematopoietic progenitors. The Proportion of JAK2 T Allele Patients GNC T Allele (%) Expanded Cells T Allele (%) PV1 (Female) 81 10 PV2 (Male) 77 28 PV3 (Male) 44 42 PV4 (Female) 78 19 PV5 (Female) 78 28

scholarly journals Conditional expression of heterozygous or homozygous Jak2V617F from its endogenous promoter induces a polycythemia vera–like disease

Blood ◽  
2010 ◽  
Vol 115 (17) ◽  
pp. 3589-3597 ◽  
Author(s):  
Hajime Akada ◽  
Dongqing Yan ◽  
Haiying Zou ◽  
Steven Fiering ◽  
Robert E. Hutchison ◽  
...  
Keyword(s):  
Polycythemia Vera ◽  
Gene Dosage ◽  
Myeloproliferative Neoplasms ◽  
Primary Myelofibrosis ◽  
Erythroid Progenitors ◽  
Conditional Expression ◽  
Biochemical Analyses ◽  
Genetic Events ◽  
Serum Erythropoietin

Abstract A somatic point mutation (V617F) in the JAK2 tyrosine kinase was found in a majority of patients with polycythemia vera (PV), essential thrombocythemia, and primary myelofibrosis. However, contribution of the JAK2V617F mutation in these 3 clinically distinct myeloproliferative neoplasms (MPNs) remained unclear. To investigate the role of JAK2V617F in the pathogenesis of these MPNs, we generated an inducible Jak2V617F knock-in mouse, in which the expression of Jak2V617F is under control of the endogenous Jak2 promoter. Expression of heterozygous mouse Jak2V617F evoked all major features of human polycythemia vera (PV), which included marked increase in hemoglobin and hematocrit, increased red blood cells, leukocytosis, thrombocytosis, splenomegaly, reduced serum erythropoietin (Epo) levels and Epo-independent erythroid colonies. Homozygous Jak2V617F expression also resulted in a PV-like disease associated with significantly greater reticulocytosis, leukocytosis, neutrophilia and thrombocytosis, marked expansion of erythroid progenitors and Epo-independent erythroid colonies, larger spleen size, and accelerated bone marrow fibrosis compared with heterozygous Jak2V617F expression. Biochemical analyses revealed Jak2V617F gene dosage-dependent activation of Stat5, Akt, and Erk signaling pathways. Our conditional Jak2V617F knock-in mice provide an excellent model that can be used to further understand the molecular pathogenesis of MPNs and to identify additional genetic events that cooperate with Jak2V617F in different MPNs.

scholarly journals Combination of BCL-2 Inhibition and Triptolide Causes Synthetic Lethality in Acute Myeloid Leukemia through Preventing Reciprocal Resistance

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2083-2083
Author(s):  
Bing Xu ◽  
Yuanfei Shi ◽  
Long Liu ◽  
Bing Z Carter
Keyword(s):  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Synthetic Lethality ◽  
Leukemic Cells ◽  
Apoptotic Pathway ◽  
P53 Status ◽  
Acute Myeloid

BCL-2 inhibition exerts effective pro-apoptotic activities in acute myeloid leukemia (AML) but clinical efficacy as a monotherapy was limited in part due to the treatment-induced MCL-1 increase. Triptolide (TPL) exhibits anti-tumor activities in part by upregulating pro-apoptotic BCL-2 proteins and decreasing MCL-1 expression in various malignant cells. We hypothesized that combined BCL-2 inhibition and TPL exert synergistic anti-leukemia activities and prevent the resistance to BCL-2 inhibition in AML. We here report that TPL combined with BCL-2 inhibitor ABT-199 synergistically induced apoptosis in leukemic cells regardless of p53 status through activating the intrinsic mitochondrial apoptotic pathway in vitro. Although ABT-199 or TPL alone inhibited AML growth in vivo, the combination therapy demonstrated a significantly stronger anti-leukemic effect. Mechanistically, TPL significantly upregulated BH3 only proteins including PUMA, NOXA, BID and BIM and decreased MCL-1 but upregulated BCL-2 expression in both p53 wild type and p53 mutant AML cell lines, while the combination decreased both BCL-2 and MCL-1 and further increased BH3 only BCL-2 proteins. MCL-1 and BCL-2 increases associated with respective ABT-199 and TPL treatment and resistance were also observed in vivo. Significantly downregulating MCL-1 and elevating BH3 only proteins by TPL could not only potentially block MCL-1-mediated resistance but also enhance anti-leukemic efficacy of ABT-199. Conversely, BCL-2 inhibition counteracted the potential resistance of TPL mediated by upregulation of BCL-2. The combination further amplified the effect, which likely contributed to the synthetic lethality. This mutual blockade of potential resistance provides a rational basis for the promising clinical application of TPL and BCL-2 inhibition in AML independent of p53 status. Disclosures Carter: Amgen: Research Funding; AstraZeneca: Research Funding; Ascentage: Research Funding.

scholarly journals Epidemiology of Patients with Classical Philadelphia-Chromosome Negative Myeloproliferative Neoplasms at a Single Academic Medical Center in Singapore

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 5478-5478
Author(s):  
Priscella Shirley Chia ◽  
Vanessa CL Chong ◽  
Ting Yuan Tay ◽  
Eng Soo Yap ◽  
Wee Joo Chng ◽  
...  
Keyword(s):  
Southeast Asia ◽  
Ethnic Groups ◽  
Research Funding ◽  
Myeloproliferative Neoplasms ◽  
Local Population ◽  
Philadelphia Chromosome ◽  
Leukemic Transformation ◽  
Female Ratio ◽  
Platelet Counts ◽  
The Mean

Abstract Introduction: Myeloproliferative Neoplasms (MPNs) represents a disorder that involves abnormal proliferation of cells originating from the myeloid line. The proliferation of these cells can lead to complications that are at times fatal. Despite its potential to cause life threatening complications, there is little data on this disease in Southeast Asia. As Singapore is a multiracial country in Southeast Asia, there may be some disease characteristics exclusive to patients here due to its unique population composition. The data from this Southeast Asian cohort would be useful to determine disease homogeneity in Asian countries. Methods: A retrospective review of the MPN database from National University Hospital, Singapore (NUHS) revealed 320 patients who were clinically diagnosed with MPN from 2008 to 2017. This data included patients with Essential Thrombocythemia (ET), Polycythemia Rubra Vera (PRV), Primary Myelofibrosis (PMF), Myeloproliferative Neoplasm, unclassifiable (MPN-U) and Chronic Eosinophilic Leukemia (CEL) (Figure 1A) as per the 2017 WHO classification. For this analysis, we included only the classical Philadelphia chromosome negative MPN and focused on the epidemiology, transformation and overall survival rate. Results: There was a slight male predominance with a male to female ratio of 1.3:1. The ethnic groups within this cohort consisted of 65.8% ethnic Chinese, 20.7% Malay, 5.5% Indian and the rest were made up of other ethnic groups within the region such as Eurasians, Thai, Filipinos, Burmese, Indonesians, Bangladeshi, Vietnamese and Arabian patients. The mean age at diagnosis for this group was 60.5. The mean age was 59.2 years for ET, 61.2 for PRV and the mean age of PMF was the oldest at 63.8. The mean age of diagnosis for ET and PMF patients in our cohort was slightly older compared to the Korean cohort (55.4 and 59.5 years) (Byun, et al., 2016). The majority of this cohort was made up of ET patients (53.1%) followed by PRV (35.3%) and PMF (11.6%). 77.5% of these patients were JAK2 V617F mutation (JAK2) positive. The percentage of patients who were JAK2 positive for ET, PRV and PMF were 69.2%, 96.9% and 56.3% respectively. The percentage of JAK2 positive patients for the three subtypes were higher in our local population compared to the Chinese and Japanese cohorts. Only 120 patients were tested for Calreticulin Exon 9 (CALR) mutations as this molecular test was only available in our institution from 2015 onwards. ET patients make up 68.4% of CALR positive patients. It was noted that CALR positive patients had comparatively higher mean platelet counts of 925.2 than CALR negative patients with mean platelet counts of 691.7. This phenomenon is seen in both CALR positive ET and CALR positive MF patients. In the 10-year period, 25 patients were lost to follow up and 8 patients transferred their care to another institution. Overall, 27 patients were deceased, with a mean survival of 3.5 years. The death-to-case ratio was 11.5 per 100 cases. The death-to-case ratio for ET, PRV and PMF is 6.1 per 100, 8.2 per 100 and 31.3 per 100 respectively. During this period, only 6 patients had transformation. Three patients progressed to post-ET myelofibrosis and 3 had leukemic transformation. Those who had leukemic transformation were initially diagnosed with PRV (1 patient) and PMF (2 patients). All patients who had leukemic transformation were deceased and had a mean survival of 1.4 years from the transformation event. Conclusion: Whilst there were some observable differences between our data and existing Asian data, there is still insufficient information to determine disease homogeneity. This is partly due to the rapid growth of molecular knowledge in this field and the regular revision of the WHO diagnostic criteria of MPNs over the last decade or so. There needs to be coordinated efforts within the region to ensure that our patients have equal access to these diagnostic platforms and that they receive an accurate diagnosis. Disclosures Chng: Janssen: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding; Aslan: Research Funding; Merck: Research Funding; Takeda: Consultancy, Honoraria, Other: Travel, accommodation, expenses; Celgene: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding; Amgen: Consultancy, Honoraria, Other: Travel, accommodation, expenses.

scholarly journals Targeting mTORC1/2 by a mTOR Kinase Inhibitor (PP242) induces Apoptosis In AML Cells Under Conditions Mimicking Bone Marrow Microenvironment

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 778-778
Author(s):  
Zhihong Zeng ◽  
Yuexi Shi ◽  
Twee Tsao ◽  
Yihua Qiu ◽  
Steven M. Kornblau ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Mouse Model ◽  
Signaling Pathways ◽  
Research Funding ◽  
Kinase Inhibitor ◽  
Annexin V ◽  
Mtor Signaling ◽  
Leukemic Cells ◽  
Survival Signaling

Abstract Abstract 778 The prognosis of patients with acute myeloid leukemia (AML) remains poor. Our studies have demonstrated that chemoresistance of AML is not solely due to increased survival signaling in AML cells, but is also enhanced by microenvironment/leukemia interactions. Bone marrow-derived mesenchymal cells (MSC) comprise an essential component of the leukemia bone marrow microenvironment. MSC have the capacity to support normal and malignant hematopoiesis and protect leukemic cells from chemotherapy. We have previously reported that co-culture of AML cells with MSC results in activation of multiple pro-survival signaling pathways in leukemic cells, from which phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling is the key upstream regulator of survival and chemoresistance (Tabe et al., 2007 Cancer Res. 2007). In this study, we investigated the role of mTOR signaling in primary AML cells co-cultured with stroma and in the in vivo leukemia mouse model utilizing a novel TOR kinase inhibitor PP242 (Intellikine, La Jolla, CA). Unlike rapamycin and its analogs, which suppress TORC1 only partially and do not acutely inhibit TORC2, PP242 has been reported to achieve greater inhibition of both TOR complexes, resulting in broader suppression of the PI3K/AKT/TOR signaling in Ph+ B-ALL and T-cell lymphoma (Feldman, et al., PLoS Biol 2009; Janes, et al., Nat Med. 2010). We first employed reverse phase protein array (RPPA) technique profiling of 53 proteins to determine the changes in activation of signaling pathways in leukemic cells from 20 primary AML samples co-cultured with murine stromal line MS-5. Co-culture with stroma resulted in activation of multiple signaling pathways in primary AML cells, inducing upregulation of pAKT(Thr308) in 18, mTOR in 17, pERK(Thr202/204) in 14, and pSTAT3(Ser727) in 12 of the 20 pt samples. This resulted in significant decrease of spontaneous apoptosis in primary AML samples (average 33.7 ± 3.8% annexin V(+) cells in primary AML without co-culture vs. 19.6 ± 3.1% in primary AML co-cultured with MS5, p = 0.027, n = 20). In a next set of experiments, blockade of mTOR signaling with PP242, in a dose dependent fashion, effectively induced apoptosis in primary AML samples (n = 9) cultured with or without stroma: at 60nM, 6.4 ± 1.8% and 8.8 ± 2.4% specific apoptosis (annexin V+), respectively; at 190nM, 10.5% ± 52.8% and 14.9% ± 3.9%; at 560nM, 17.6.9 ± 5.7%; and 21.9 ± 4.9% at 1.67uM, 27.2 ± 6.1% and 27.3 ± 5.8%; at 5uM, 38.8 ± 6.5% and 37.1 ± 7.2%. Importantly, at low nanomolar concentrations, PP242 attenuates the activities of both TORC1 and TORC2, resulting in inhibition of phosphorylation of AKT at S473, S6K at S240/244 and 4EBP1 at T37/46 in both, primary AML cells and most importantly in MSC cultured alone or co-cultured with AML. In the in vivo leukemia mouse model utilizing GFP/luc-labeled Baf3-FLT3/ITD cells, PP242 (60mg/kg/QD gavage) exerted significantly greater anti-leukemia activity compared with TORC1 inhibitor rapamycin (0.1mg/kg/QD IP, p = 0.03). PP242 suppressed leukemia progression as determined by bioluminescence imaging (average luminescence intensity 5.65 ± 1.75 in control vs. average 2.75 ± 0.65 in PP242 group) and significantly extended survival (p = 0.005). In summary, our findings indicate a novel therapeutic strategy to target leukemia within the BM microenvironment through efficient blockade of mTOR/AKT signaling with novel selective TORC kinase inhibitor. This research is funded by Intellikine. Disclosures: Liu: Intellikine: Employment. Rommel:Intellikine: Employment. Fruman:Intellikine: Research Funding. Konopleva:Intellikine: Research Funding.

scholarly journals Histone H3K36me2-specific methyltransferase ASH1L is required for the MLL-AF9-induced leukemogenesis

2021 ◽  
Author(s):  
Mohammad B. Aljazi ◽  
Yuen Gao ◽  
Yan Wu ◽  
George I. Mias ◽  
Jin He
Keyword(s):  
Enzymatic Activity ◽  
Transcriptional Activation ◽  
Gene Knockout ◽  
Conditional Knockout ◽  
Leukemic Cells ◽  
Gene Promoters ◽  
Leukemic Transformation ◽  
Knockout Mouse Model

ASH1L and MLL1 are two histone methyltransferases that facilitate transcriptional activation during normal development. However, the roles of ASH1L and its enzymatic activity in the development of MLL-rearranged leukemias are not fully elucidated in the Ash1L gene knockout animal models. In this study, we used an Ash1L conditional knockout mouse model to show that loss of ASH1L in hematopoietic progenitor cells impaired the initiation of MLL-AF9-induced leukemic transformation in vitro. Furthermore, genetic deletion of ASH1L in the MLL-AF9-transformed cells impaired the maintenance of leukemic cells in vitro and largely blocked the leukemia progression in vivo. Importantly, the loss of ASH1L function in the Ash1L-deleted cells could be rescued by wild-type but not the catalytic-dead mutant ASH1L, suggesting the enzymatic activity of ASH1L was required for its function in promoting MLL-AF9-induced leukemic transformation. At the molecular level, ASH1L enhanced the MLL-AF9 target gene expression by directly binding to the gene promoters and modifying the local histone H3K36me2 levels. Thus, our study revealed the critical functions of ASH1L in promoting the MLL-AF9-induced leukemogenesis, which provides a molecular basis for targeting ASH1L and its enzymatic activity to treat MLL-arranged leukemias.

scholarly journals Platelet-Dependent Thrombin Generation Induced By ADP or Activated Factor X Does Not Contribute to Increased In Vivo Thrombin Formation in Myeloproliferative Neoplasms

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4336-4336
Author(s):  
Christina Berens ◽  
Heiko Rühl ◽  
Jens Müller ◽  
Johannes Oldenburg ◽  
Peter Brossart ◽  
...  
Keyword(s):  
Plasma Levels ◽  
Research Funding ◽  
Thrombin Generation ◽  
Myeloproliferative Neoplasms ◽  
Final Concentration ◽  
Factor X ◽  
Prothrombinase Complex ◽  
Significant Difference ◽  
Thrombin Formation

Abstract Introduction: Myeloproliferative Neoplasms (MPN), including the clinical entities Polycythemia Vera (PV), Essential Thrombocythemia (ET), and Primary Myelofibrosis (PMF), are characterized by an increased thrombotic risk, the pathomechanisms of which are not well-understood. It has been suggested that an increased sensitivity of platelets to adenosin diphosphate (ADP) contributes to the hypercoagulable state in PV and ET through increased thrombin generation. In the present study we analyzed plasma levels of thrombin and platelet-dependent thrombin generation in MPN patients with an additional focus on prothrombin activation by the prothrombinase complex. Methods: A total of 33 blood samples were obtained from patients with MPN (PV, n=18; ET, n=5; PMF, n=10) and from 33 healthy blood donors that served as controls. In vitro thrombin generation in platelet-rich plasma (PRP) and platelet-poor plasma (PPP) was assessed using the Calibrated Automated Thrombogram (CAT) assay. To induce thrombin generation either ADP (1 µmol/L final concentration) or activated factor X (FXa, 10 ng/mL final concentration) were applied. To further characterize the MPN-associated hypercoagulable state in vivo, plasma levels of free thrombin were measured using an oligonucleotide-based enzyme capture assay (OECA). Prothrombin activation fragment 1+2 (F1+2), thrombin-antithrombin complex (TAT), and D-dimer were measured additionally. Results: In PRP of MPN patients a slightly higher ADP-induced peak thrombin concentration (Cpeak) was observed than in the healthy controls, with 106 (79-130) vs. 84 (65-110) nmol/L (median and interquartile range, p=.026). There was no statistically significant difference in the ADP-induced endogenous thrombin potential (ETP) in MPN patients (1445, 1194-1643 nmol/L·min) compared with the controls (1417, 1258-1814 nmol/L·min). There was no statistically significant difference in the FXa-induced Cpeak and ETP between MPN patients and controls, with 106 (79-127) vs. 97 (82-128) nmol/L, and 1424 (1165-1560) vs. 1641 (1193-1841) nmol/L·min, respectively. With 0.68 (<0.46-1.20) pmol/L, plasma levels of free thrombin were significantly higher (p=.025) in MPN patients than in the control group, in which median thrombin levels were below the limit of detection. Plasma levels of F1+2 and TAT were also higher in the MPN group than in healthy controls, with 0.31 (0.17-0.50) vs. 0.18 (0.13-0.25) nmol/L (p=.002) and 4.36 (2.53-6.76) vs. 2.36 (<2.00-2.68) ng/mL (p=.003), respectively. Conclusion: Increased plasma levels of thrombin, F1+2, and TAT indicate enhanced in vivo thrombin formation in MPN patients. Despite a slightly increased ADP sensitivity of MPN-platelets, the total amount of thrombin generated in PRP from MPN patients is not increased. This makes it unlikely that a 'hyperreactivity' of MPN platelets, resulting in increased activities of the prothrombinase complex on the platelet surface, contributes to the increased thrombin formation in MPN patients. Disclosures Berens: Shire: Research Funding; Biotest: Research Funding; Pfizer: Research Funding; Sanofi Genzyme: Research Funding; CSL-Behring: Research Funding. Rühl:Shire: Research Funding; Swedish Orphan Biovitrum: Consultancy, Research Funding; Grifols: Research Funding; Sanofi Genzyme: Research Funding; CSL-Behring: Research Funding. Müller:Swedish Orphan Biovitrum: Consultancy, Research Funding. Oldenburg:Roche: Honoraria, Research Funding; Grifols: Honoraria, Research Funding; Chugai: Honoraria, Research Funding; Novo Nordisk: Honoraria, Research Funding; Bayer: Consultancy, Honoraria, Research Funding; Shire: Honoraria, Research Funding; Octapharma: Honoraria, Research Funding; CSL Behring: Honoraria, Research Funding; Biogen: Honoraria, Research Funding; Biotest: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Swedish Orphan Biovitrum: Honoraria, Research Funding.

CREB Regulates Early Myelopoiesis and Myeloid Engraftment.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1452-1452
Author(s):  
Tiffany Simms-Waldrip ◽  
Michelle Yoonha Cho ◽  
Kenneth Dorshkind ◽  
Kathleen M Sakamoto
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Cell Proliferation ◽  
Research Funding ◽  
Myeloid Cells ◽  
Myeloid Cell ◽  
Wild Type ◽  
In Vivo Models ◽  
Hematopoietic Stem

Abstract Abstract 1452 The cAMP-responsive element binding protein (CREB) is a nuclear transcription factor that regulates genes that control cell proliferation, differentiation, and survival. CREB overexpression leads to increased proliferation and survival of myeloid cells. Transgenic (Tg) mice overexpressing CREB under the control of the myeloid specific promoter hMRP8 develop myeloproliferative disease (MPD) but not leukemia. We hypothesized that transplantation of hematopoietic stem cells from CREB transgenic mice into lethally irradiated recipient wild type mice would lead to enhanced myelopoiesis and myeloid engraftment. The goal of our study was to determine if proliferative stress through transplantation would result in increased myeloid engraftment and progression of CREB overexpressing cells from MPD to leukemia. Steady state analyses were performed on CREB Tg mice, including flow cytometry to resolve common myeloid progenitors (CMP), granulocyte macrophage progenitors (GMP), and megakaryocyte erythroid progenitors (MEP), as well as cell cycle analysis to determine baseline proliferative state. In vitro and in vivo models that exposed CREB-expressing cells to proliferative stress were used. In the former case, long-term bone marrow cultures (LTBMC) were established on an adherent layer of stromal cells prepared from wild type (WT) bone marrow (BM) with media specific for myeloid cell growth. BM cells (2 × 106) from CREB Tg mice or WT controls were seeded onto the stroma and evaluated at 4 and 8 weeks for myeloid cell proliferation. In vivo studies were conducted by transplanting (2.5 × 106) BM cells from CREB Tg mice into lethally irradiated recipients that were sacrificed at 4 weeks. Cells harvested from LTBMC or transplant recipients were analyzed by flow cytometry to evaluate cell lineage and proliferation or were plated in methylcellulose and assessed for colony formation. In addition, kinetic analyses were performed on these populations. At baseline, CREB Tg mice have an increased percentage of early progenitors (1.8% vs. 1.2%, p=0.0001) with increased absolute numbers of CMP (17,683 cells vs. 11,650 cells, p=0.0001) at 12 weeks of age compared to WT controls. CREB Tg mice also have increased number of cells in S phase at baseline (26% vs. 20%, p=0.0022) due to upregulation of cyclins A and D. LTBMCs seeded with BM cells from CREB Tg mice had greater numbers of myeloid cells at 4 weeks compared to cultures established with WT marrow (4.5 × 106 cells/mL and 1.3 × 106 cells/mL respectively, p = 0.0135). Consistent with these data, mice transplanted with CREB Tg BM had a significantly higher percentage of donor myeloid cells at 4 weeks, detected using cell surface markers Gr-1+Mac-1+ (67% vs. 40%, p=0.0061). These mice also had a higher percentage of more differentiated Mac-1+ myeloid cells (11% vs. 0%, p=0.0014) and a higher number of myeloid cells in BM colony assays compared to recipients of WT marrow (69% vs. 13%, p<0.0001). At 4 weeks post-transplant, the histology of the spleen and liver from mice transplanted with CREB Tg marrow demonstrated replacement of the lymphocytes in the white pulp with macrophages, as well as extramedullary hematopoiesis in the liver that was not observed in WT controls. Our results provide evidence that CREB overexpression enhances myelopoiesis and short-term myeloid engraftment, but is not sufficient for transformation to AML. Therefore, CREB plays a critical role in normal hematopoietic dynamics and myeloid progenitor cell kinetics. Disclosures: Sakamoto: Abbott Laboratories, Inc.: Research Funding; Genentech, Inc.: Research Funding.

Metabolic Capability to Induce the Pasteur Effect Mediates Sensitivity of Human Leukemic Cells to Metformin

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2601-2601
Author(s):  
Sarah Scotland ◽  
Estelle Saland ◽  
Lindsay Peyriga ◽  
Rémi Peyraud ◽  
Elizabeth Micklow ◽  
...  
Keyword(s):  
Cell Lines ◽  
Research Funding ◽  
Leukemic Cell ◽  
Leukemic Cells ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Pasteur Effect ◽  
Cytotoxic Response ◽  
Leukemic Cell Lines

Abstract Abstract 2601 An emerging hallmark of cancer cells is the reprogramming of intermediary and energy metabolism these cells undergo. Several epidemiological studies have shown that metformin, widely used to treat patients with type 2 diabetes, may reduce their risk of cancer. Despite several reports of anti-neoplastic activity of metformin, the mechanisms responsible for this activity have not been fully elucidated in cancer or leukemic cells. We hypothesized that metformin elicits a metabolic reprogramming driven by alterations in mitochondrial function and signaling, which induces apoptosis in leukemic cells, and that metabolic flexibility determines the variation(s) of the cytotoxic response to metformin among different leukemic cell lines. We first demonstrated that metformin markedly decreased oxygen consumption of six leukemic cell lines in a concentration-dependent manner. We also observed that the cytotoxic effect of metformin varies between cell lines reflecting their energetic capacity to compensate for the mitochondrial inhibition induced by metformin (eg. to induce the Pasteur effect). Importantly, metformin-insensitive leukemic cells did not exhibit a Pasteur effect in response to metformin. All leukemic cells exhibited high basal conversion of glucose to lactate (eg. aerobic glycolysis) and specific expression of key metabolic genes as compared to normal mononuclear cells. Despite dependence on glucose catabolism, metformin sensitivity was associated with relative resistance to glucose starvation. Metformin effects in drug-resistant cells were potentiated by the addition of a glycolytic inhibitor, but not by inhibitors of the pentose phosphate pathway or glutaminolysis. Leukemic cells with broad metabolic capacities to utilize other energetic substrates in response to diverse nutrient starvation showed insensitivity to metformin. Metformin induced a significant decrease in metabolites of the upper segment of glycolysis and the oxidative branch of the pentose phosphate pathway as well as a clear increase of PRPP and IMP biosynthesis. Energy charge, the nucleotide phosphate pool and lactate/glucose ratio remained stable after metformin treatment. Furthermore, our results showed that basal glucose uptake/consumption and the activity of the lower segment of the glycolytic pathway are key determinants of a cytotoxic response to metformin. In addition, high glutathione, malate, IMP and orotate content were observed in metformin-insensitive leukemic cells. Moreover, the cytotoxic effect of metformin was independent of AMPK/LKB1 status of the leukemic cells while p53 expression abrogated this effect. The presence of wild-type p53 appears to partially protect tumor cells from glucose starvation and metformin cytotoxicity and prevents the induction of the Pasteur effect. Finally, we demonstrated that metformin increased the cytotoxicity of chemotherapy agent, cytarabine, on all leukemic cell lines in vitro and significantly reduced leukemic colony-forming units (CFU-L) from six primary AML patient samples in a concentration-dependent manner. Additional experiments on metabolic and signaling pathways as well as in vivo studies are in progress to better understand the cytotoxic response of metformin in both AML cell lines and primary AML patient specimens that impact the therapeutic potential of metformin in vivo. Disclosures: Carroll: Agios Pharmaceuticals: Research Funding; TetraLogic Pharmaceuticals: Research Funding; Sanofi Aventis Corporation: Research Funding; Glaxo Smith Kline, Inc.: Research Funding.

Sign in / Sign up

Export Citation Format

Share Document