SHIP1 and PTEN Co-Inactivation and Akt Pathway Targeting in ALL.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2386-2386
Author(s):  
Mitchell B. Diccianni ◽  
Lisa M. Barnhill ◽  
Tony Lo ◽  
Alice L. Yu

Abstract Abstract 2386 Poster Board II-363 Despite success in the treatment of Acute lymphoblastic leukemia (ALL), relapse and death continue to occur and in survivors, secondary malignancies due to the impact of aggressive chemotherapy negatively impact the quality and duration of life. Akt plays a central role in signal transduction and is negatively regulated by PTEN and, in cells of hematopoietic lineage, SHIP1; it is positively regulated by PIK3CA. Despite multiple levels of regulation, Akt is aberrantly activated in many cancers including ALL. To understand the mechanism behind Akt activation in ALL, PIK3CA, PTEN and SHIP1 genes were assessed in leukemia cell lines and primary samples. No sample harbored PIK3CA mutation. PTEN was expressed in just one-third of the cell lines analyzed, but in two-thirds of the primary ALL of both T- and B-lineage, though all were in the phosphorylated (inactivated) form. SHIP1 was expressed in all leukemia cell lines, except for Jurkat and K562 cell lines. In general, expression of SHIP1 protein was much higher in cell lines of the B-lineage than of the T-lineage. The Jurkat T-ALL cell line, long observed to lack SHIP1 protein, harbored biallelic inactivation by null mutation and a frame-shift deletion; no SHIP1 mutations were identified in the K562 cell line. In contrast to leukemia cell lines, no full length SHIP1 was detected in primary T-ALL, and only ∼20% of the primary B-precursor ALL expressed full length SHIP despite the expression of full length transcript. Both ALL lineages expressed truncated isoforms of SHIP, which cloning and sequence analysis revealed was due to premature termination resulting from the frame-shift and other translationally-inactivating alternative splicing. The co-inactivation of SHIP and PTEN may create therapeutic opportunities. The PI3KCA inhibitor LY294002 has been shown to sensitize glioblastoma and colon carcinoma to doxorubicin. To determine if these agents acted synergistically in T-ALL, cell lines Molt16 (SHIP1+/PTEN-), Jurkat (SHIP1-/PTEN-) and normal MNCs (SHIP1+/PTEN+) were subjected to various combinations of the PIK3CA inhibitor LY294002 and doxorubicin and cell proliferation and viability assessed. Normal cells were generally insensitive to both agents except at high concentrations. LY294002 and doxorubicin inhibited viability in Molt16 with IC50s of 10 uM and 0.1 ug/ml, respectively, with no evidence of synergy. Jurkat cells exhibited a similar IC50 to LY294002, but were more sensitive to doxorubicin (IC50s of 10 uM and 0.025 ug/ml, respectively), with the two agents demonstrating some synergy when added simultaneously at low concentrations. In summary, we present evidence of Akt pathway deregulation in T-ALL due to SHIP1/PTEN inactivation and suggest that agents targeting this pathway may provide new directions into the therapeutic treatment of T-ALL. Disclosures: No relevant conflicts of interest to declare.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4110-4110
Author(s):  
Yuping Gong ◽  
Xi Yang ◽  
Ting Niu

Abstract Abstract 4110 Objective To study the proliferative inhibition of imatinib, daunorubicin and bortezomib in two kinds of Ph(+) leukemia cell lines: chronic myelogenous leukemia cell line K562 expressing P210 protein and acute lymphoblastic leukemia cell line SUP-B15 expressing P190 protein. Methods (1) Cell proliferation with imatinib, daunorubicin and bortezomib for 72 hours was analyzed by the MTT assay and displayed by growth curve and IC50 value. (2) The change of bcr-abl gene mRNA levels after the 48 hours' intervention of imatinib (final concentration at 0μM, 0.35μM, 1 μM) was detected by reverse transcription polymerase chain reaction (RT-PCR). Results (1) The IC50 values of K562 and SUP-B15 cells inhibited by imatinib, daunorubicin and bortezomib for 72 hours was respectively 0.286±0.06 (μmol/L), 0.303±0.009 (μmol/L), 22.127±3.592 (nmol/L) and 1.387±0.180(μmol/L), 0.117±0.017 (μmol/L), 12.350±0.740 (nmol/L), which indicated that the K562 cell line was the more sensitive to imatinib than SUP-B15 cell line, whereas the SUP-B15 cell line had the more sensitivity to daunorubicin and bortezomib. (2) There was no change of bcr-abl gene expression after the 48 hours' intervention of imatinib in both cell lines. Conclusion (1) Imatinib, daunorubicin and bortezomib had good anti-cancer effect to Ph+ leukemia cells in vitro. What's more, the K562 cell was the more sensitive to imatinib and only imatinib will have good effect on chronic myelogenous leukemia. Whereas the SUP-B15 cell had the more sensitivity to daunorubicin and bortezomib and combining imatinib with daunorubicin or bortezomib, the effect will be better on Ph(+) acute lymphoblastic leukemia. (2) The short time intervention of imatinib had no effect on the bcr-abl gene expression and imatinib could need long time to show curative effect for the Ph+ leukemia. Disclosures: No relevant conflicts of interest to declare.


1994 ◽  
Vol 14 (11) ◽  
pp. 7604-7610
Author(s):  
H M Pomykala ◽  
S K Bohlander ◽  
P L Broeker ◽  
O I Olopade ◽  
M O Díaz

Interstitial deletions of the short arm of chromosome 9 are associated with glioma, acute lymphoblastic leukemia, melanoma, mesothelioma, lung cancer, and bladder cancer. The distal breakpoints of the deletions (in relation to the centromere) in 14 glioma and leukemia cell lines have been mapped within the 400 kb IFN gene cluster located at band 9p21. To obtain information about the mechanism of these deletions, we have isolated and analyzed the nucleotide sequences at the breakpoint junctions in two glioma-derived cell lines. The A1235 cell line has a complex rearrangement of chromosome 9, including a deletion and an inversion that results in two breakpoint junctions. Both breakpoints of the distal inversion junction occurred within AT-rich regions. In the A172 cell line, a tandem heptamer repeat was found on either side of the deletion breakpoint junction. The distal breakpoint occurred 5' of IFNA2; the 256 bp sequenced from the proximal side of the breakpoint revealed 95% homology to long interspersed nuclear elements. One- and two-base-pair overlaps were observed at these junctions. The possible role of sequence overlaps, and repetitive sequences, in the rearrangement is discussed.


Leukemia ◽  
1997 ◽  
Vol 11 (3) ◽  
pp. 441-447 ◽  
Author(s):  
CC Uphoff ◽  
RAF MacLeod ◽  
SA Denkmann ◽  
TR Golub ◽  
A Borkhardt ◽  
...  

Blood ◽  
1998 ◽  
Vol 92 (5) ◽  
pp. 1497-1504 ◽  
Author(s):  
Zhu-Gang Wang ◽  
Roberta Rivi ◽  
Laurent Delva ◽  
Andrea König ◽  
David A. Scheinberg ◽  
...  

Abstract Inorganic arsenic trioxide (As2O3) and the organic arsenical, melarsoprol, were recently shown to inhibit growth and induce apoptosis in NB4 acute promyelocytic leukemia (APL) and chronic B-cell leukemia cell lines, respectively. As2O3 has been proposed to principally target PML and PML-RAR proteins in APL cells. We investigated the activity of As2O3 and melarsoprol in a broader context encompassing various myeloid leukemia cell lines, including the APL cell line NB4-306 (a retinoic acid–resistant cell line derived from NB4 that no longer expresses the intact PML-RAR fusion protein), HL60, KG-1, and the myelomonocytic cell line U937. To examine the role of PML in mediating arsenical activity, we also tested these agents using murine embryonic fibroblasts (MEFs) and bone marrow (BM) progenitors in which the PML gene had been inactivated by homologous recombination. Unexpectedly, we found that both compounds inhibited cell growth, induced apoptosis, and downregulated bcl-2 protein in all cell lines tested. Melarsoprol was more potent than As2O3 at equimolar concentrations ranging from 10−7 to 10−5 mol/L. As2O3 relocalized PML and PML-RAR onto nuclear bodies, which was followed by PML degradation in NB4 as well as in HL60 and U937 cell lines. Although melarsoprol was more potent in inhibiting growth and inducing apoptosis, it did not affect PML and/or PML-RAR nuclear localization. Moreover, both As2O3 and melarsoprol comparably inhibited growth and induced apoptosis of PML+/+ and PML−/− MEFs, and inhibited colony-forming unit erythroid (CFU-E) and CFU granulocyte-monocyte formation in BM cultures of PML+/+ and PML−/− progenitors. Together, these results show that As2O3 and melarsoprol inhibit growth and induce apoptosis independent of both PML and PML-RAR expression in a variety of myeloid leukemia cell lines, and suggest that these agents may be more broadly used for treatment of leukemias other than APL. © 1998 by The American Society of Hematology.


Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1488-1488 ◽  
Author(s):  
Ebenezer David ◽  
Rajni Sinha ◽  
Claire Torre ◽  
Jonathan L. Kaufman ◽  
Sagar Lonial

Abstract Introduction: Novel agents as anti-cancer therapy are used in the setting of specific molecular abnormalities that provide a survival advantage for malignant cells. One such agent, tipifarnib, is theoretically targeted at Ras mutations which are present in a number of different human cancers. Our previous experience with the FTIs (David et al, in press Blood) has demonstrated that they are ideal agents to combine with other targeted agents. We have investigated the combination of the AKT inhibitor perifosine with tipifarnib in human leukemia and lymphoma cell lines with the hypothesis that the combination of 2 targeted agents will disrupt separate survival pathways and ultimately result in synergistic tumor cell death. Methods: In this study we used the human leukemia cell lines HL-60, Jurkat, and the lymphoma cell line HT. Western blot analysis was used to assess for the effect of either single agent perifosine, tipifarnib, or the combination on AKT, p-AKT, PDK-1, and caspase cleavage. Flow cytometry was utilized to assess for Annexin V staining following combination therapy. Results:Dose escalation studies demonstrated that doses of tipifarnib up to 5μm demonstrated a significant cell death in HL-60 and HT cells. Perifosine doses of 1–5uM also induced cell death in both HL-60 and HT cells. When apoptosis was assessed using western blot analysis of caspase 3 activity and cleavage, the combination of perifosine and tipifarnib demonstrated significant apoptosis using low doses of both agents. The apoptosis was associated with downregulation of phos-PDK1, with a resultant downregulation in p-AKT. The level of phos-PDK1 was completely inhibited in less than 24 hrs in both the HL-60 and HT cell lines in combination than when either agent was given alone. Conclusion: The combination of perifosine, and AKT targeted agent, with tipifarnib, a Ras targeted agent, appear to induce significant cell death in lymphoma and leukemia cell lines with rapid downregulation of p-AKT via the PDK-1 pathway. This apoptosis occurs in vitro using concentrations well below those that have been achieved in current clinical trials using these agents. Additional studies are being carried out to further delineate the mechanism of synergy as well as to further explore the impact of sequence of administration using this combination. Further studies are also planned to xplore the impact of the combination on primary human leukemia and lymphoma cells from the blood and bone marrow.


Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1986-1986
Author(s):  
Xuejun Zhang ◽  
Li Wen ◽  
Fuxu Wang ◽  
Ling Pan ◽  
Jianmin Luo ◽  
...  

Abstract Tumor Necrosis factor (TNF)-related apoptosis- inducing ligand (TRAIL) is a new member of TNF superfamily discovered recently. Several studies showed that TRAIL can preferentially induce apoptosis in a variety of tumor cells, while most normal cells tested do not appear to be sensitive to TRAIL. In the present study, we treated K562 and U937 leukemia cell lines with recombinant mutant human TRAIL (rmhTRAIL) alone or together with daunorubicin (DNR) to investigate the apoptosis of the treated cells and the synergistic reaction of rmhTRAIL and DNR. The normal cell line MRC-5 was used as control. The expression of four TRAIL receptors mRNA (death receptor DR4 and DR5, decoy receptor DcR1 and DcR2) in the cells lines were detected before and after the treatment by DNR. (1) AO-EB double staining and TUNEL staining were used to evaluate the morphological change of leukemia cell lines before and after the treatment. The results showed that rmhTRAIL could induce the apoptosis of leukemia cell lines and a dose-dependent manner was found in leukemia cell lines but not in MRC-5 cell lines. (2) The growth inhibition rate of leukemia cell lines induced by rmhTRAIL alone or combined with DNR was examined with MTT assays. Different concentrations of rmhTRAIL(8, 40, 200, 1000ng/mL)alone or combined with DNR(8, 40, 200, 1000ng/mL) was used. The result showed a dose-dependent growth inhibition by rmhTRAIL alone for K562- and U937-cell line (P<0.05) also, but not for MRC-5 cell line (P>0.05). The IC50 for K562 cells and for U937 cells had no statistic difference (538.80 vs 301.56ng/mL, P>0.05). In leukemia cell lines, the growth inhibition rates in combination groups were much higher than in rmhTRAIL or DNR alone groups (P<0.05), and no synergistic killing effects was found in MRC-5 cells (P<0.05). It was concluded that rmhTRAIL had synergistic effects with DNR in the growth inhibition of K562 and U937 cells. (3). To explore the antitumor mechanisms of rmhTRAIL combined with DNR, the expression level of the DR4, DR5 and DcR1, DcR2 mRNA in these three cell lines was examined by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) before and after the treatment with DNR. The high expression of DR4,DR5 mRNA in the tested cells were observed before the treatment of DNR, while very low or even undetectable expression level of DcR1 and DcR2 mRNA were observed in U937 and K562 cells, and a high expression level of DcR1 and DcR2 mRNA in MRC-5 cells were observed. After 24 hours treatment of three cell lines with DNR (200ng/ml), the expression level of DR5 mRNA increased in K562 and U937 cells (P<0.05). DR4 mRNA also increased in K562 cells but not in U937 cells. There was no change in DcR1 and DcR2 mRNA level in three cell lines. The four receptors’ mRNA level in MRC-5 cells was not influenced by DNR. Our results indicated that rmhTRAIL could induce the apoptosis of leukemia cell lines, and DNR could enhance significantly the sensitivity of K562 and U937 cells to apoptosis induced by rmhTRAIL through up-regulation of death receptors. Therefore, we presumed TRAIL might be act as a new agent for biological therapy in leukemia.


Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4726-4726
Author(s):  
David W. Rusnak ◽  
Sharon K. Rudolph ◽  
Afshin Safavi ◽  
Connie L. Erickson-Miller

Abstract Abstract 4726 The thrombopoietin receptor agonists (TPO-RA), romiplostim and eltrombopag, are presently indicated for the treatment of certain patient groups with immune thrombocytopenia purpura. In a clinical study with romiplostim in patients with low-/intermediate-1 risk myelodysplastic syndromes (MDS), cases of transient increases in blast cell counts were observed and cases of MDS disease progression to acute myeloid leukemia (AML) were reported. In the present study, we evaluated the impact of romiplostim, eltrombopag, and recombinant human thrombopoietin (TPO) on the proliferation of 5 human AML and 1 TPO-dependent megakaryoblastic cell line. The cell lines evaluated include the TPO-dependent cell line, N2C TPO; the TPO-R positive AML lines, HEL92.1.7 and OCI-AML-3; and the TPO-R negative AML cell lines, HL60, THP-1, and NOMO-1. All cells were exposed to 11-point dose response curves of the 3 agents at concentrations sufficient to generate a full stimulatory response in the N2C TPO cell line. Cells were exposed to concentrations of romiplostim and eltrombopag that met or exceeded the reported Cmax achieved for each agent in high-dose clinical trials and were 3- (eltrombopag) to 30-fold (romiplostim) above trough levels from the same clinical trials. Neither romiplostim nor TPO treatment resulted in detectable stimulation or inhibition of leukemia cell growth at concentrations up to 10 μg/mL. Treatment with eltrombopag up to 40 μg/mL caused inhibition of all AML cell lines with mean IC50 values ranging from 6.4 to 13.5 μg/mL. These IC50 values reflect concentrations that are 3- to 6-fold below the Cmax of a 300 qd dose of eltrombopag (40.5 μg/mL) and at concentrations as low as 2-fold below Ctau levels (12.4 μg/mL). Cmax exceeded the IC90 for these AML cell lines, which ranged from 18.5 to 27.9 μg/mL. No stimulation of AML growth was evident through the range of the eltrombopag dose response curve on any of the cell lines evaluated. The results of this study confirm earlier in vitro studies (Will 2009, Erickson-Miller 2010) showing inhibitory effects of eltrombopag on leukemic cell lines and support clinical studies to evaluate a potential anti-leukemic effect of higher doses of eltrombopag in patients with AML. Disclosures: Rusnak: GlaxoSmithKline: Consultancy, Equity Ownership, Patents & Royalties. Off Label Use: Eltrombopag is an oral TPO agonist indicated for chronic ITP being studied in MDS/AML. Rudolph:GlaxoSmithKline: Consultancy, Equity Ownership. Erickson-Miller:GlaxoSmithKline: Employment, Equity Ownership, Patents & Royalties, Research Funding.


Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2230-2230 ◽  
Author(s):  
Marcus M Schittenhelm ◽  
Figen Akmut ◽  
Barbara Illing ◽  
Julia Frey ◽  
Katja Schuster ◽  
...  

Abstract Activating mutations of the class III receptor tyrosine kinases FLT3 and KIT are associated with certain human neoplasms, including hematologic malignancies, i.e. the majority of patients with systemic mast cell disorders (KIT) and subsets of patients with acute myelogenous leukemia (FLT3 and KIT). Crenolanib is a potent selective FLT3 inhibitor with high efficacy against internal tandem dupliction mutations (ITD) – but also secondary kinase domain mutations conferring resistance towards other TKI. Interestingly, crenolanib does not target the wildtype KIT isoform, which is believed to reduce clinical side effects such as prolonged myelosuppression observed with other TKI. Clinical studies are currently enrolling. We now show that gain-of-function mutations of KIT, including codon D816 alterations as the most prevalent mutation in SM and CBFL, sensitize the mutant isoform to crenolanib. Several mast cell and leukemia cell lines harboring autoactivating KIT or FLT3 isoforms were treated with crenolanib in dose dilution series (MOLM14, MV4;11, HMC1.1/1.2, p815). To minimize cell-type specific off-target effects, an isogenic cell model was established. The murine pro B-cell line Ba/F3 was retrovirally transduced with either a FLT3 ITD or a KIT D816 isoform. Apoptosis induction was analyzed by annexin V-based assays. FLT3/KIT tyrosine phosphorylation was assessed by western immunoblots. As previously described, the FLT3 ITD positive cell line MOLM14 revealed high sensitivity towards crenolanib with IC50s in the lowest nanomolar range. We also confirmed high sensitivity towards crenolanib ex vivo in the low nanomolar range in a native sample of a heavily pretreated patient. This patient relapsed with FLT3 ITD positive leukemia harboring a secondary D835H mutation in a subclone. Interestingly, leukemia cells in the relapse situation were much more oncogene-addicted than cells at primary diagnosis, which is in line with previous findings by others. Due to the structural homology of FLT3 D835 and KIT D816 mutations, we extended our studies to mutant-KIT mastocytosis and leukemia cell models and confirm clinically relevant antiproliferative as well as proapoptotic sensitivities towards crenolanib: for HMC mastocytosis cells harboring a KIT V560G and/or a D816V mutation, potent induction of apoptosis was observed with IC50s of 100-250nM. The murine p815 mastocytosis cell line (harboring a D814Y mutation corresponding to D816Y in humans) demonstrated a proapoptotic effect of crenolanib with an IC50 of 60 nM. Treatment of corresponding KIT or FLT3 isoform-transduced Ba/F3 cells confirmed similar IC50s in the leukemia cell lines. Parental Ba/F3cells did not show any sensitivity towards crenolanib up to concentrations of 1000 nM. Additionally, potent dephosphorylation at 100 nM of KIT D816V in Ba/F3 and HMC cells after exposure to crenolanib confirmed mutant-KIT as a target of the drug. Evaluation of a broader range of native mast cell and leukemia patient samples as well as additional leukemia cell lines and isogenic Ba/F3 KIT or FLT3 transfectants is ongoing. First results demonstrate activity of crenolanib in native cells of a subset of patient samples with SM or CBFL treated ex vivo. Even more, combination of crenolanib with anthracyclines revealed additive to superadditive proapoptotic effects. Moreover, combination of crenolanib with cladribine, a hallmark agent in the treatment of systemic mastocytosis, resulted in potent induction of apoptosis already at doses that did not display any proapoptotic effects when administered as single agents, thereby providing a rationale for combinatorial therapeutic approaches. In summary, crenolanib is effective against the KIT D816V isoform associated with several hematologic malignancies. Notably, while not as effective towards mutant-KIT compared to the FLT3 ITD isoform, the observed estimated IC50 of crenolanib is well in the range of achievable plasma concentrations and in the range of the potent KIT inhibitor dasatinib, which is successfully under clinical investigation in CBFL. Our data provide a rationale to test crenolanib as a potent inhibitor of mutant-KIT isoforms in KIT-associated neoplasms. Disclosures Schuster: AROG Pharmaceuticals: Employment. Ramachandran:AROG: Employment.


Blood ◽  
1998 ◽  
Vol 92 (5) ◽  
pp. 1497-1504 ◽  
Author(s):  
Zhu-Gang Wang ◽  
Roberta Rivi ◽  
Laurent Delva ◽  
Andrea König ◽  
David A. Scheinberg ◽  
...  

Inorganic arsenic trioxide (As2O3) and the organic arsenical, melarsoprol, were recently shown to inhibit growth and induce apoptosis in NB4 acute promyelocytic leukemia (APL) and chronic B-cell leukemia cell lines, respectively. As2O3 has been proposed to principally target PML and PML-RAR proteins in APL cells. We investigated the activity of As2O3 and melarsoprol in a broader context encompassing various myeloid leukemia cell lines, including the APL cell line NB4-306 (a retinoic acid–resistant cell line derived from NB4 that no longer expresses the intact PML-RAR fusion protein), HL60, KG-1, and the myelomonocytic cell line U937. To examine the role of PML in mediating arsenical activity, we also tested these agents using murine embryonic fibroblasts (MEFs) and bone marrow (BM) progenitors in which the PML gene had been inactivated by homologous recombination. Unexpectedly, we found that both compounds inhibited cell growth, induced apoptosis, and downregulated bcl-2 protein in all cell lines tested. Melarsoprol was more potent than As2O3 at equimolar concentrations ranging from 10−7 to 10−5 mol/L. As2O3 relocalized PML and PML-RAR onto nuclear bodies, which was followed by PML degradation in NB4 as well as in HL60 and U937 cell lines. Although melarsoprol was more potent in inhibiting growth and inducing apoptosis, it did not affect PML and/or PML-RAR nuclear localization. Moreover, both As2O3 and melarsoprol comparably inhibited growth and induced apoptosis of PML+/+ and PML−/− MEFs, and inhibited colony-forming unit erythroid (CFU-E) and CFU granulocyte-monocyte formation in BM cultures of PML+/+ and PML−/− progenitors. Together, these results show that As2O3 and melarsoprol inhibit growth and induce apoptosis independent of both PML and PML-RAR expression in a variety of myeloid leukemia cell lines, and suggest that these agents may be more broadly used for treatment of leukemias other than APL. © 1998 by The American Society of Hematology.


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