FLT3-ITD Enhances Proliferation and Survival of AML Cells through Activation of RSK1 to Upregulate the mTORC1/eIF4F Pathway Cooperatively with PIM or PI3K and to Inhibit Bad and Bim

FLT3-ITD is the most frequent tyrosine kinase mutation in acute myeloid leukemia (AML) associated with poor prognosis. We previously reported that FLT3-ITD activates the mTORC1/S6K/4EBP1 pathway cooperatively through the STAT5/PIM and PI3K/Akt pathways in AML cells, thus promoting proliferation and survival as well as therapy resistance at least partly by enhancing the eIF4F complex-mediated cap-dependent translation of growth- and survival-related mRNAs. The RSK family serine/threonine protein kinases, including the predominant isoforms expressed in AML RSK1 and RSK2, posses the N- and C-terminal kinase domains (NTKD and CTKD); CTKD is activated by Erk and recruits PDK1 to activate NTKD, which phosphorylates downstream substrates to promote cell growth and survival through regulation of translation as well as transcription. Although RSK2 was reported to be activated by FLT3-ITD or upregulated through PIM and to be required for leukemogenesis by FLT3-ITD but not by BCR/ABL, these studies examined only activation of CTKD, used mainly the CTKD inhibitor FMK, and did not adequately examine the downstream signaling mechanisms from RSK. Thus, we examined the significance of RSK in FLT3-ITD AML and the underlying molecular mechanisms. The RSK NTKD inhibitors LJI308 and LJH685 as well as FMK distinctively inhibited FLT3-ITD-dependent proliferation of the AML cell line MV4-11 and model hematopoietic cell lines, whereas BCR/ABL-dependent proliferation of various cells were resistant. Furthermore, inhibition of FLT3-ITD by quizartinib or gilteritinib, but not that of BCR/ABL by imatinib, prominently inhibited RSK1 and RSK2 NTKDs, as examined by the activation-specific phosphorylation in their T-loop sites, associated with inhibition of the MEK/Erk pathway and PDK1 in MV4-11. Inhibition of FLT3-ITD also reduced expression of RSK1 but not RSK2, which, however, was not mimicked by inhibition of PIM. Intriguingly, FMK or LJI308 inhibited FLT3-ITD and phosphorylation of its substrate STAT5 in MV4-11 cells as well as in primary AML cells expressing FLT3-ITD. By using the most specific RSK inhibitor LJH685 as well as sh-RNA-mediated knock down, CRISPR/CAS9-mediated knockout, or overexpression of RSK1 or RSK2 in MV4-11 cells, we found that mainly RSK1 but not RSK2 phosphorylates not only S6RP on S235/236 but also TSC2 on S1798 to upregulate the mTORC1/S6K/4EBP1 pathway. RSK1 also phosphorylated eIF4B on S422 and, in cooperation with PIM, on S408, which is required for efficient cap-dependent translation by the eIF4F complex. Furthermore, RSK1 phosphorylated Bad on S112, which is instrumental for dissociation from anti-apoptotic proteins Bcl-2 and Bcl-xL. On the other hand, inhibition of RSK or Erk by LJH685 or SCH772984, respectively, increased the expression level of BimEL in the S65-phosphorylated or unphosphorylated form, respectively, which is consistent with the idea that RSK phosphorylates BimEL after its phosphorylation on S65 by Erk to induce proteasomal degradation of this potent pro-apoptotic protein. Interestingly, inhibition of RSK upregulated the MEK/Erk pathway, which correlated with increased binding of SHP2 with Gab2 phosphorylated by FLT3-ITD and was prevented by the SHP2 inhibitor IIB-08, thus revealing the negative feed back mechanisms involving the Gab2/SHP2 complex. LJH685 or RSK1 knockout reduced viable cell numbers of MV4-11 as well as FLT3-ITD-positive primary AML cells synergistically with the PIM inhibitor AZD-1208 or the PI3K inhibitor GDC-0941 at least partly by inducing apoptosis, which was prevented by overexpression of Bcl-xL or Mcl-1. Knock down of mTOR in MV4-11 cells also enhanced apoptosis induced by inhibition of PIM or PI3K. Moreover, LJH685 sensitized MV4-11 significantly to BH3 mimetics, such as venetoclax or A-1210477, which inhibits Bcl-2 or Mcl-1, respectively. Our findings reveal that FLT3-ITD but not BCR/ABL activates RSK1 as well as RSK2 and upregulates RSK1 expression to promote proliferation and survival of leukemic cells through upregulation of the mTORC1 pathway and modification of eIF4B in collaboration with the PI3K/Akt and STAT5/PIM pathways and additionally through inhibitory modification of the proapoptotic proteins Bad and Bim (Figure). Thus, RSK1 represents a promising molecular target particularly in combination with PIM or PI3K for novel therapeutic strategies against therapy-resistant FLT3-ITD-positive AML. Disclosures No relevant conflicts of interest to declare.

Download Full-text

A Key Role for Tumor Suppressor BTG1 In the Regulation of Metabolic Stress Responses: Implications for Asparaginase Resistance In Pediatric Acute Lymphoblastic Leukemia.

Blood ◽

10.1182/blood.v116.21.1028.1028 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1028-1028

Author(s):

Laurens van der Meer ◽

Marloes Levers ◽

Liesbeth van Emst ◽

Joost C. van Galen ◽

Ad Geurts van Kessel ◽

...

Keyword(s):

Acute Lymphoblastic Leukemia ◽

Stress Responses ◽

Molecular Mechanisms ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Metabolic Stress ◽

Therapy Resistance ◽

Metabolic Enzyme ◽

Central Component ◽

Pediatric Acute Lymphoblastic Leukemia

Abstract Abstract 1028 Background: L-Asparaginase (ASNase) is a key component of multi-agent chemotherapy regimens used in the treatment of pediatric Acute Lymphoblastic Leukemia (ALL). This bacterially-derived enzyme lowers blood asparagine levels by catalyzing the hydrolysis of L-asparagine, leading to specific killing of leukemic blasts. However, relapses and associated therapy resistance occur in about 20% of the patients. Although the molecular mechanisms that contribute to ASNase resistance are not well understood, increased expression of asparagine synthethase (ASNS), the enzyme responsible for asparagine synthesis, has been linked to ASNase resistance both in ALL cell lines and in primary leukemias. We recently reported that micro-deletions affecting the B-cell translocation gene 1 (BTG1) occur in about 10% of pediatric pre-B ALL cases. Here we show that BTG1 loss contributes to ASNase resistance by up-regulation of ASNS. Results: Using RNA interference, we show that loss of BTG1 expression promotes cell growth and renders pre-B ALL cells completely refractory to ASNase induced cell death. Resistance to ASNase in the BTG1 knockdown cells is accompanied by increased expression of ASNS, while knockdown of this metabolic enzyme is sufficient to reverse therapy resistance, indicating that upregulation of ASNS is required for the observed resistance phenotype. We further show that BTG1 associates with and regulates the activity of the transcription factor ATF4, a key regulator of metabolic stress responses and a central component in the regulatory network that controls ASNS expression. Conclusion: Together, our experiments identify BTG1 as an important regulator of ASNS expression which acts by modulating ATF4 function. We expect that a detailed molecular understanding of how BTG1 loss contributes to ASNase resistance, will lead to the identification of pharmacological targets that can be used to improve treatment responses in therapy-resistant ALL. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Genome-Wide shRNA Screen Implicates Mitogen Activated Protein Kinase (MAPK) Pathway In Glucocorticoid Resistance In Acute Lymphoblastic Leukemia

Blood ◽

10.1182/blood.v122.21.1296.1296 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1296-1296

Author(s):

Jones L. Courtney ◽

Christy M. Gearheart ◽

Fosmire Susan ◽

Wang Jinhua ◽

Danielle S. Bitterman ◽

...

Keyword(s):

Acute Lymphoblastic Leukemia ◽

Molecular Mechanisms ◽

Conflicts Of Interest ◽

Mapk Pathway ◽

Lymphoblastic Leukemia ◽

Mitogen Activated Protein Kinase ◽

Leukemic Cells ◽

P Value ◽

Sequencing Analysis ◽

Increased Sensitivity

Abstract Resistance to glucocorticoids (GC) is a hallmark of relapsed acute lymphoblastic leukemia (ALL) and is a predictor of outcome at diagnosis. In spite of the importance of GC in the treatment of ALL and other hematological malignancies the molecular mechanisms that lead to effective eradication of leukemic cells is incompletely understood. To address this problem we have performed a functional screen for genes involved in prednisolone resistance in ALL cell line (Reh) and correlated these results with our previously published results using an integrated genomic analysis to discover genes (pathways) altered at relapse (Hogan et al 2011). Cells were infected with a pooled whole genome shRNA library that contained approximately 80,000 shRNAs targeting 18,000 genes. Deep sequencing was used to identify shRNAs enriched or depleted upon treatment with prednisolone. Three computational methods including; bioinformatics for next generation sequencing analysis (BiNGS), redundancy & fold change analysis (RFC) and strict standardized mean difference (SSMD) were applied to the sequencing data in efforts to obtain the most robust set of candidate genes for validation (Porter et. al, Leukemia 2012, Zhang XD, J Biomol Screen, 2007). Through our primary screen a total of 263 genes were identified to modulate prednisolone sensitivity in ALL. Upon knockdown, 142 genes increased the sensitivity of the cells to prednisolone and 121 genes increased resistance to prednisolone. Five genes overlapped with genes previously identified to be altered at relapse compared to matched diagnosis samples including SLC6A18, AARSD1, MIER3, CDC42BPB, and YAP1. We hypothesize that genes that are altered at relapse in ALL and identified through functional genomics screening to modulate chemosensitivity in vitro, are likely drivers of chemoresistance and eventual relapse. We also performed gene ontology (GO) analysis using DAVID Bioinformatics to identify pathways that may be responsible for altered resistance to prednisolone. This analysis strongly implicated the mitogen-activated kinase (MAPK) pathway. The MAPK was also identified as a pathway with increased activity at relapse through our integrative genomics analysis(Hogan et al 2011). One gene of particular interest was MAP2K4 which encodes for MEK4, an upstream kinase involved in JNK phosphorylation and c-Jun activation. Knockdown of MAP2K4 by shRNA in B-precursor ALL cell lines (Reh and RS4;11) results in statistically significant (p-value < 0.5) increased sensitivity to prednisolone induced apoptosis at a range of prednisolone concentrations but not to other chemotherapy tested (etoposide, doxorubicin, and 6-thioguanine).604. Molecular Pharmacology, Drug Resistance: Poster I Upon treatment with prednisolone MAP2K4 knockdown cells have increased levels of prednisolone responsive genes GILZ (1.4-2.4 fold in Reh, 2.1-3.8 fold in RS4;11) and TXNIP (1.8-5.7 fold in Reh, 2.5-2.6 in RS4;11). Increased sensitivity to prednisolone and increased levels of prednisolone responsive genes was associated with decreased levels of p-JNK that has been previously implicated in regulating glucocorticoid signaling through phosphorylation of the glucocorticoid receptor (GR) at S226 (Roatsky et. al, PNAS 1998, Itoh et. al, Mol. Endo. 2002).s Overall this data suggests that decreased levels of MAP2K4 results in increased sensitivity to GC by increasing GC signaling and implicates MEK4 as novel drug target in ALL. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

FLT3-ITD Confers Resistance to Bortezomib By Protecting the mTOR/4EBP1 Pathway through Activation of STAT5 and Induction of Pim-1 Expression

Blood ◽

10.1182/blood.v126.23.1270.1270 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1270-1270

Author(s):

Ayako Nogami ◽

Keigo Okada ◽

Gaku Oshikawa ◽

Shinya Ishida ◽

Hiroki Akiyama ◽

...

Keyword(s):

Molecular Mechanisms ◽

Kinase Inhibitor ◽

Conflicts Of Interest ◽

Expression Level ◽

Apoptotic Pathway ◽

Expression Levels ◽

Combined Use ◽

Significant Difference ◽

Induced Apoptosis ◽

Novel Therapeutic Strategies

Abstract FLT3-ITD and FLT3-TKD are the most frequent tyrosine kinase mutations in AML, with the former strongly associated with a poor prognosis. We have recently revealed that FLT3-ITD confers resistance to the PI3K/AKT pathway inhibitors by protecting the mTOR/4EBP1/Mcl-1 pathway through STAT5 activation in AML. The proteasomal inhibitor bortezomib (BZM) has recently been reported as a promising agent for treatment of AML. We examine here the molecular mechanisms involved in induction of apoptosis by BZM in hematopoietic cells, including AML cells, and evaluate the effects of FLT3-ITD and TKD on these mechanisms and on the sensitivity of cells to BZM-induced apoptosis. We first comparatively examined the effect of BZM on survival of hematopoietic 32D cells and human leukemic UT7 cells driven by FLT3-ITD (32D/ITD and UT7/ITD) or FLT3-D835Y (32D/TKD and UT7/TKD). BZM induced activation of Bax, decline in mitochondrial membrane potential, and activation of caspase-9, thus leading to apoptosis, more conspicuously in cells driven by FLT3-ITD than FLT3-TKD. These results indicate that FLT3-ITD, as compared with FLT3-TKD, conferred resistance to apoptosis induced by BZM through the intrinsic pathway in these cells. To elucidate the molecular mechanisms involved in BZM-induced apoptosis in these cells, we next examined the effect of BZM on expression levels of FLT3 in these cells as well as in human leukemic MV4-11 cells expressing FLT3-ITD. Intriguingly, treatment of these cells with BZM conspicuously reduced expression levels of FLT3. It was further reveled that BZM further facilitated the decline in FLT3-ITD expression in MV4-11 cells treated with actinomycin D to shut down the transcriptional activity. Furthermore, BZM significantly retarded the recovery of FLT3-ITD expression in MV4-11 cells washed out from the translation inhibitor cycloheximide. These results suggest that BZM may downregulate the FLT3-ITD expression mainly at the translational level. However, we did not observe any significant difference in extent of the BZM-induced decline in expression levels between FLT3-ITD and FLT3-TKD. We next examined the effect of BZM on the mTOR/4EBP1 pathway, which we have shown to play important roles in regulation of apoptosis downstream of FLT3-ITD. It was found that BZM downregulated this pathway more significantly in 32D/TKD cells than in 32D/ITD cells. Because we have also previously found that STAT5 activated robustly by FLT3-ITD plays an important role in modulation of the mTOR/4EBP1 pathway, we examined the effects of BZM on 32D/TKD cells forced to express the constitutively activated STAT5 mutant, STAT5A1*6. As expected, STAT5A1*6 conferred resistance to BZM-induced downregulation of the mTOR/4EBP1 pathway as well as apoptosis in 32D/TKD cells. Consistent with this, the STAT5 inhibitor pimozide, clinically in use for neuropsychiatric disorders, abrogated the resistance of 32D/ITD, UT7/ITD, and MV4-11 cells to BZM-induced inhibition of the mTOR/4EBP1 pathway and apoptosis. We finally examined the possible involvement of the STAT5 target gene product Pim-1 in acquisition of resistance to BZM by cells expressing FLT3-ITD. We fist confirmed that Pim-1 was expressed at a higher level in 32D/ITD cells than in 32D/TKD cells and that STAT5A1*6 increased the expression level of Pim-1 in 32D/TKD cells. We then examined the effects of a specific Pim kinase inhibitor, AZD-1208, and found that it synergistically downregulated the mTOR/4EBP1 pathway and induced apoptosis with BZM in 32D/ITD cells as well as 32D/TKD cells expressing STAT5A1*6. We also examined the effects of a BET bromodomain inhibitor, JQ1, which has recently been shown to inhibit the STAT5 activity and to reduce specifically the expression level of Pim-1 as well as c-Myc in MV4-11 cells. As expected, pretreatment of 32D/ITD or MV4-11 cells with JQ1 conspicuously sensitized these cells to BZM-induced apoptosis. These results suggest that BZM downregulates FLT3 expression and the mTOR/4EBP1 pathway to activate the intrinsic apoptotic pathway and that robust STAT5 activation by FLT3-ITD confers resistance to BZM on AML cells through protection of the mTOR/4EBP1 pathway at least partly by inducing Pim-1 expression. The present study may contribute to development of novel therapeutic strategies against FLT3-ITD-positive AML by combined use of BZM and the STAT5/Pim-1 pathway inhibitors. Disclosures No relevant conflicts of interest to declare.

Download Full-text

EphA3 As a Target For Monoclonal Antibody Therapy For Acute Leukemia

Blood ◽

10.1182/blood.v122.21.5013.5013 ◽

2013 ◽

Vol 122 (21) ◽

pp. 5013-5013 ◽

Cited By ~ 1

Author(s):

Sara Charmsaz ◽

Kirrilee J Miller ◽

Bryan W Day ◽

Fares El-Ajeh ◽

Geoff T Yarranton ◽

...

Keyword(s):

Monoclonal Antibody ◽

Bone Marrow ◽

Conflicts Of Interest ◽

Xenograft Model ◽

High Expression ◽

Leukemic Cells ◽

Control Group ◽

Acute Leukemias ◽

Knock Down ◽

Vascular Elements

Abstract Eph receptor tyrosine kinases interact with cell-surface ephrin ligands to direct cell growth, migration and cell positioning. High expression of EphA3 in a number of cancers has been linked to progression through facilitation of invasiveness and metastatic spread. EphA3 protein, which was originally described in leukemia, has also reported to be expressed in sarcomas, lung cancer, melanoma and glioblastoma. Using a Humaneered® derivative of the IIIA4 anti-EphA3 monoclonal antibody, KB004 (KaloBios Pharmaceuticals Inc), a clinical trial has commenced which targets the EphA3 protein in leukemia. The EphA3-specific monoclonal antibody, IIIA4, binds and activates human and mouse EphA3 with similar affinities, binding is followed by internalization of receptor-antibody complexes. We have shown high expression of EphA3 in the LK63 pre-B ALL and no expression of EphA3 in the Reh, a similar pre-B ALL cell line. We have examined the effect of the IIIA4 antibody in LK63 and Reh NOD/SCID xenograft models. In the LK63 xenograft model, administration of the IIIA4 antibody led to inhibition of tumour growth and decrease spread from bone marrow to the spleen and other organs and increased the latency of the disease, however no reduction in engraftment was observed in Reh xenograft model, suggesting that the effect was directed against the leukemic cells rather than the stromal and vascular elements. To further analyse the function of EphA3 expression, LK63 EphA3 knock down and EphA3-transfected Reh cells were tested in the xengraft model. Similar to LK63, antibody-treated Reh EphA3 xenografts showed reduction in the bone marrow engraftment and increased the latency of the disease. In contrast, LK63 EphA3 knock down xenografts showed minimal difference between treated and control group but notably showed a significant reduction in the splenic engraftment compared to the normal LK63 model. These results are consistent with IIIA4-mediated effects on the growth of EphA3-positive leukemic cells in the bone marrow and a secondary effect on the subsequent spread of the leukemic cells to extra-medullary sites and provides evidence that EphA3 is a functional therapeutic target in acute leukemias. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

FLT3-ITD Activates RSK1 to Enhance Proliferation and Survival of AML Cells by Activating mTORC1 and eIF4B Cooperatively with PIM or PI3K and by Inhibiting Bad and BIM

Cancers ◽

10.3390/cancers11121827 ◽

2019 ◽

Vol 11 (12) ◽

pp. 1827 ◽

Cited By ~ 4

Author(s):

Watanabe ◽

Nogami ◽

Okada ◽

Akiyama ◽

Umezawa ◽

...

Keyword(s):

Myeloid Leukemia ◽

Feedback Regulation ◽

Therapeutic Strategies ◽

Erk Pathway ◽

Negative Feedback Regulation ◽

Inhibition Of Proliferation ◽

Eif4f Complex ◽

Promising Target ◽

Novel Therapeutic Strategies ◽

Kinase Mutation

FLT3-ITD is the most frequent tyrosine kinase mutation in acute myeloid leukemia (AML) associated with poor prognosis. We previously found that FLT3-ITD activates the mTORC1/S6K/4EBP1 pathway cooperatively through the STAT5/PIM and PI3K/AKT pathways to promote proliferation and survival by enhancing the eIF4F complex formation required for cap-dependent translation. Here, we show that, in contrast to BCR/ABL causing Ph-positive leukemias, FLT3-ITD distinctively activates the serine/threonine kinases RSK1/2 through activation of the MEK/ERK pathway and PDK1 to transduce signals required for FLT3-ITD-dependent, but not BCR/ABL-dependent, proliferation and survival of various cells, including MV4-11. Activation of the MEK/ERK pathway by FLT3-ITD and its negative feedback regulation by RSK were mediated by Gab2/SHP2 interaction. RSK1 phosphorylated S6RP on S235/S236, TSC2 on S1798, and eIF4B on S422 and, in cooperation with PIM, on S406, thus activating the mTORC1/S6K/4EBP1 pathway and eIF4B cooperatively with PIM. RSK1 also phosphorylated Bad on S75 and downregulated BIM-EL in cooperation with ERK. Furthermore, inhibition of RSK1 increased sensitivities to BH3 mimetics inhibiting Mcl-1 or Bcl-2 and induced activation of Bax, leading to apoptosis, as well as inhibition of proliferation synergistically with inhibition of PIM or PI3K. Thus, RSK1 represents a promising target, particularly in combination with PIM or PI3K, as well as anti-apoptotic Bcl-2 family members, for novel therapeutic strategies against therapy-resistant FLT3-ITD-positive AML.

Download Full-text

Receptor Activator of NF-κB (RANK) Confers Resistance to Chemotherapy in AML and Associates with Dismal Disease Course

Cancers ◽

10.3390/cancers13236122 ◽

2021 ◽

Vol 13 (23) ◽

pp. 6122

Author(s):

Kim Clar ◽

Lisa Weber ◽

Bastian Schmied ◽

Jonas Heitmann ◽

Maddalena Marconato ◽

...

Keyword(s):

Molecular Mechanisms ◽

Ex Vivo ◽

Treatment Options ◽

Tumor Necrosis Factor Receptor ◽

Surface Expression ◽

Leukemic Cells ◽

Disease Course ◽

Growth And Survival ◽

Receptor Activator ◽

Cytostatic Compounds

Although treatment options of acute myeloid leukemia (AML) have improved over the recent years, prognosis remains poor. Better understanding of the molecular mechanisms influencing and predicting treatment efficacy may improve disease control and outcome. Here we studied the expression, prognostic relevance and functional role of the tumor necrosis factor receptor (TNFR) family member Receptor Activator of Nuclear Factor (NF)-κB (RANK) in AML. We conducted an experimental ex vivo study using leukemic cells of 54 AML patients. Substantial surface expression of RANK was detected on primary AML cells in 35% of the analyzed patients. We further found that RANK signaling induced the release of cytokines acting as growth and survival factors for the leukemic cells and mediated resistance of AML cells to treatment with doxorubicin and cytarabine, the most commonly used cytostatic compounds in AML treatment. In line, RANK expression correlated with a dismal disease course as revealed by reduced overall survival. Together, our results show that RANK plays a yet unrecognized role in AML pathophysiology and resistance to treatment, and identify RANK as “functional” prognostic marker in AML. Therapeutic modulation of RANK holds promise to improve treatment response in AML patients.

Download Full-text

Comparative Clinical and Biological Features of Primary Mediastinal B-Cell Lymphoma (PMBL) and Mediastinal Grey Zone Lymphoma (MGZL).

Blood ◽

10.1182/blood.v114.22.106.106 ◽

2009 ◽

Vol 114 (22) ◽

pp. 106-106 ◽

Cited By ~ 8

Author(s):

Kieron Dunleavy ◽

Stefania Pittaluga ◽

Kevin Tay ◽

Nicole Grant ◽

Clara C Chen ◽

...

Keyword(s):

B Cell ◽

Fdg Pet ◽

Molecular Mechanisms ◽

Conflicts Of Interest ◽

B Cell Lymphoma ◽

Significant Benefit ◽

Grey Zone ◽

Mediastinal Radiation ◽

Related Group ◽

Novel Therapeutic Strategies

Abstract Abstract 106 Mediastinal B-cell lymphomas (MBCL) are a clinical-pathologically related group, putatively derived from thymic B-cells, and include PMBL, Nodular Sclerosis Hodgkin Lymphoma (NSHL) and MGZL. PMBL and NSHL contain significant molecular overlap, and MGZL has pathological features intermediate between PMBL and NSHL. Overlapping clinical features include young age, female predominance and localized mediastinal presentation. The risk of local failure after anthracycline-based therapy has led to routine mediastinal radiation (RT) in PMBL and bulky NSHL, suggesting relative chemo-resistance. Anecdotal reports also suggest MGZL is relatively resistant to Hodgkin-based chemotherapy. Thus, better systemic therapy is needed to avoid RT and its risk of secondary malignancies and cardiac disease, and to improve cure. Clinical studies suggest PMBL and NSHL are particularly responsive to dose-intense strategies. We prospectively compared untreated PMBL and MGZL for the first time and assessed if the dose-intense DA-EPOCH-R regimen would obviate the need for RT and improve cure. DA-EPOCH-R was administered 6-8 cycles and FDG-PET was used in pts with residual masses to assess need for biopsy and RT (n=31). PMBL and MGZL had similar clinical characteristics shown below. PMBL and MGZL IHC, respectively, had similar CD20+ 100% both; CD10+ 5% and 0%; BCL6+ 86% and 100%; and MUM1+ 58% and 67%; but different CD30+ 71% and 100% (P=0.14) and CD15+ 0% and 60% (P=0.0009). At 4 years median F/U, PFS and OS were 100% for patients with PMBL without RT (table below). Comparison to historical PMBL (n=17) treated with DA-EPOCH (PFS 65% and OS 77% at 10 years) indicated a significant benefit of rituximab (PFS: P=0.0012) and (OS: P=0.013). MGZL outcome with DA-EPOCH-R was significantly worse than PMBL (PFS 30% and OS 83% at 4 years) and 50% required mediastinal RT (figure below). FDG-PET + and - predictive value for relapse were 62% and 95%, respectively. In conclusion, DA-EPOCH-R is highly effective in PMBL and obviates RT whereas MGZL shows greater chemo-resistance. Rituximab adds significant benefit to PMBL. We hypothesize that MGZL and NSHL are more chemo-resistant than PMBL and may share molecular mechanisms of drug resistance. Novel therapeutic strategies, such as targeting the microenvironment, are needed in these diseases. Comparative gene expression profiling is underway. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Coating of Tumor Cells by Platelets Confers Expression of Immunoregulatory Molecules Which Impair NK Cell Anti-Tumor Reactivity.

Blood ◽

10.1182/blood.v114.22.2993.2993 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2993-2993

Author(s):

Theresa Placke ◽

Hans-Georg Kopp ◽

Lothar Kanz ◽

Helmut R Salih

Keyword(s):

Nk Cells ◽

Tumor Cells ◽

Mhc Class I ◽

Molecular Mechanisms ◽

Nk Cell ◽

Conflicts Of Interest ◽

Class I ◽

Leukemic Cells ◽

Target Cells ◽

Tumor Immune Escape

Abstract Abstract 2993 Poster Board II-970 NK cells play an important role in cancer immunosurveillance and may prevent tumor progression and metastasis due to their ability to mediate direct cellular cytotoxicity and by releasing immunoregulatory cytokines which shape adaptive immune responses. Their reactivity is governed by various activating and inhibitory molecules expressed on target cells and reciprocal interactions with other hematopoietic cells like dendritic cells. Platelets contribute to tumor immune escape, metastasis, and angiogenesis (e.g. Jin et al Nature Med. 2006). In mice, thrombocytopenia inhibits metastasis, and this is reversed by NK cell depletion suggesting that platelets are an important additional player in NK cell-tumor interaction. However, the knowledge regarding the molecular mechanisms by which platelets influence NK cells is fragmentary at best. We found recently that platelet release soluble factors, most notably TGF-β, upon interaction with tumor cells which mediates NK cell silencing through downregulation of the activating immunoreceptor NKG2D (Kopp et al., Cancer Res 2009, in press). However, immunoregulatory molecules residing in the platelet membrane may also modulate NK cell anti-tumor responses. We report here that presence of platelets causes coating of tumor cells, and this markedly reduces NK cell lysis of tumor cells. This is mediated by conferment of “pseudoexpression” of platelet-expressed immunoregulatory molecules to tumor cells which are absent on the tumor cells alone. Among those immunregulatory molecules we identified various ligands for NK cell receptors like MHC class I, GITR ligand or CD62P. To establish the functional significance of tumor cell pseudoexpression of platelet molecules we employed functional analyses of tumor cells and NK cells with or without coating by autologous platelets. The impaired anti-tumor reactivity of NK cells against coated tumor cells was restored by blocking MHC class I on the coating platelets, while isotype control had no effect. Moreover, coating of tumor cells by platelets was validated by ex vivo analyses of primary leukemic cells from patients which also revealed substantial coating by platelets and confered expression of NK cell-modulating molecules. Our data indicate that platelets enable a molecular mimicry of tumor cells, which enables tumor cells to escape NK cell-mediated tumor immunosurveillance. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Enforced Expression of Mir-125b Blocks Granulocytic Differentiation by Simultaneous Repression of Multiple Targets.

Blood ◽

10.1182/blood.v116.21.1554.1554 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1554-1554

Author(s):

Ewa Surdziel ◽

Maciej Cabanski ◽

Iris Dallmann ◽

Arnold Ganser ◽

Michaela Scherr ◽

...

Keyword(s):

Cell Death ◽

Protein Expression ◽

Molecular Mechanisms ◽

Conflicts Of Interest ◽

Luciferase Reporter ◽

Myeloid Differentiation ◽

Granulocytic Differentiation ◽

Multiple Targets ◽

Knock Down

Abstract Abstract 1554 Granulopoiesis is a multistep process controlled by a complex system of cytokines and transcription factors that modulate expression of downstream genes and mediate proliferation and differentiation signals. Recent findings demonstrate that miRNAs may provide an additional level of control. We used the 32D murine myeloid progenitor cell line as a model system to study G-CSF-induced granulocytic differentiation. Based on miRNA-expression analyzed by microarray and miR-qRT-PCR, we identified several miRNAs (including miR-34a-c, -125b, -155, 181b, 223, 291a, 370) potentially involved in regulation of granulocytic differentiation. To define the role of individual miRNAs, stable gain- and loss-of-function phenotypes were generated using lentiviral gene transfer of pre-miRNAs and antagomiRs, respectively. We found that enforced expression of miR-125b in undifferentiated 32D myeloid precursors resulted in a complete block of granulocytic differentiation upon G-CSF treatment and partially protected the cells from IL-3 withdrawal-induced cell death. The pivotal role of miR-125b in myeloid differentiation was demonstrated in myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) with the chromosomal translocation t(2;11)(p21;q23) resulting in miR-125b over-expression [Bousquet M, et al. JEM 2008]. Furthermore, clonogenic assays of primary Lin- cells revealed that miR-125b cultures generated more and larger colonies as compared to miR-control, indicating a proliferative advantage of miR-125b over-expressing progenitor cells. Correspondingly, enforced miR-125b expression in murine bone marrow has recently been shown to induce a lethal myeloproliferative disorder (MPD) in transplanted mice [O'Connell RM, et al. PNAS 2010]. However, the molecular mechanisms mediated by miR-125b in hematopoietic cells remain largely unknown. By utilizing different miRNA-target prediction programs including Target Scan, DIANA microT v 0.3 and RNA22, we identified Bcl-2 family members Bak1, Mcl-1, Bmf and Puma as putative targets of miR-125b. We confirmed diminished Bak1 protein expression in 32D cells (reduction by ∼40%); however, lentivirus-mediated RNAi targeting Bak1 to the similar level as induced by enforced miR-125b expression resulted only in a delay of cell death in the presence of G-CSF but not in granulocytic differentiation block. Further computational analysis revealed two putative miR-125b binding sites in 3’ UTR of Stat3, the principal Stat protein activated following G-CSF treatment and robustly involved in myeloid differentiation. Specific binding of miR-125b to Stat3 3’UTR was validated by luciferase reporter assay and confirmed by western blotting (reduction by ∼30-40%). We found that shRNA-mediated strong reduction of Stat3 protein (reduction by ∼80%) enables G-CSF dependent cell proliferation (with a delay of some days), and induces a complete block of cellular differentiation. On the contrary, a mild reduction of Stat3 protein expression(by ∼30–40%), as induced by enforced expression of miR-125b, resulted only in a slight delay of cell death but not in block of granulocytic differentiation. Interestingly, simultaneous reduction of both Bak1 and Stat3 by combinatorial RNAi to similar levels as observed in the presence of enforced miR-125b expression, cooperatively delays cell death upon G-CSF treatment as compared to knock-down of Bak1 or Stat3 solely. Since miR-125b has approximately 500 predicted target genes and neither mild knock-down of Bak1 nor Stat3 alone resulted in a block of granulocytic differentiation, we may either miss an additional/unknown important target or the miR-125b-induced phenotype requires simultaneous repression of more targets in addition to Stat3 and Bak1. Nevertheless our study provides experimental evidence that miR-125-mediated phenotypes arise from mild and simultaneous down-regulation of multiple targets/signalling pathways. Therefore the precise and quantitative analysis of miRNA-targets is required to evaluate the safety and benefit of eventual miRNA-based therapeutic strategies to modulate complex cellular phenotypes. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

The Mir-17∼92 Cluster Activates mTORC1 Signaling in MCL by Targeting Multiple Regulators of the LKB1/AMPK/mTOR Pathway

Blood ◽

10.1182/blood.v120.21.5118.5118 ◽

2012 ◽

Vol 120 (21) ◽

pp. 5118-5118

Author(s):

Chengfeng Bi ◽

Chunsun Jiang ◽

Xiaoxing Jiang ◽

Xin Huang ◽

Mario R Fernandez ◽

...

Keyword(s):

Conflicts Of Interest ◽

Mammalian Target Of Rapamycin ◽

Mtor Inhibitors ◽

Mtor Pathway ◽

Akt Pathway ◽

Ampk Signaling ◽

Knock Down ◽

Protein Levels ◽

Mtorc1 Signaling ◽

Novel Therapeutic Strategies

Abstract Abstract 5118 Mantle cell lymphoma (MCL) is an aggressive hematological malignancy with a median survival ranging between 3 and 5 years. Novel therapeutic strategies are urgently needed to improve the outcome. Mammalian target of rapamycin (mTOR) pathway which plays a central role in controlling cell growth, proliferation and metabolism has been shown to be deregulated in MCL. mTOR inhibitors, such as rapamycin and its analogues, have been approved for treatment of relapsed/refractory MCL. However, the molecular mechanism of mTOR activation in MCL has yet to be defined. MiRNA (miR)-17∼92 is a cluster of six miRNAs which are frequently overexpressed in MCL cases and overexpression of the miR-17∼92 cluster in MCL predicts poor prognosis. Our previous study demonstrated that miR-17∼92 activated the PI3K/AKT pathway by directly targeting PTEN and PHLPP2; and knock-down of miR-17∼92 expression inhibited MCL tumor growth in a xenograft/SCID MCL mouse model. In the present study, we further demonstrated that knock-down miR-17∼92 decreased the cell size, similar effect as seen in cells treated with mTORC1 inhibitors. Knockdown of miR-17∼92 expression also decreased the glycolysis, protein synthesis and glucose uptake in MCL cells. We found that knockdown miR-17∼92 activated AMPK signaling as demonstrated by increased phosphorylation of AMPK at Thr172, especially under low glucose condition. Activated AMPK further phosphorylated TSC2 and Raptor at S1387 and S792, respectively, thereby inhibiting mTORC1 signaling as evidenced by decreased phosphorylation of RPS6 and 4E-BP1. Using TargetScan and other prediction algorithms, we found that several factors in the LKB1/AMPK/mTOR pathway, such as LKB1, CAB39, PRKAA1 and TSC1, are predicted the targets of miR-17∼92. In this study, we validated these factors as direct targets of the miR-17∼92 by 3'UTR luciferase assays using reporter plasmids containing the 3'UTR of the targets or the 3'UTR with mutations in the predicted miRNA binding sites. The protein levels of these targets decreased in MCL cells with the miR-17∼92 overexpression. Conversely, the levels of these factors were increased upon knockdown of miR-17∼92 cluster. Our results indicate that overexpression of miR-17∼92 in MCL plays important role in mTORC1 activation by inactivating the LKB1/AMPK signaling, in addition to its effect on the PI3K/AKT pathway activation. Disclosures: No relevant conflicts of interest to declare.

Download Full-text