Circulating CLL Cells Expressing CD49d Display a Phospho-Proteomic Profile Consistent with a Constitutive Receptor Engagement by Blood-Borne Ligands

Abstract Abstract 930 Chronic lymphocytic leukemia (CLL) cell growth and survival occur in defined microenvironment niches controlled by several receptor/ligand interactions including those mediated by the α4β1 integrin. This integrin, in particular, is known to interact with both the extracellular matrix component fibronectin, via the non-RGD CS-1 fragments, and the bone marrow/stromal components, via the vascular-cell adhesion molecule-1 (VCAM-1). The α4 integrin chain (a.k.a. CD49d) has been previously shown to be associated with poor prognosis in CLL by marking a subset of CLL patients characterized by a more aggressive clinical course both in term of disease progression and overall survival. Despite the great deal of studies investigating CLL cell microenvironmental interactions in tissue sites, little is known regarding the constitutive engagement of adhesion receptors in circulating CLL cells, and the role in this context of plasma/plasma components. To address this issue, the proteomic profiles of circulating leukemic cells from 80 CLL cases expressing or not CD49d were explored using a reverse phase protein microarray (RPMA) approach quantitatively analysing 77 proteins (61 phosphoproteins) known to be involved in translational control, cell growth, apoptosis, B cell receptor and cytoskeletal signaling. Comparison of the signaling activation portrait between 40 CD49d pos and 40 CD49d neg CLL cases highlighted the over-expression in CD49d pos CLL of proteins involved in the regulation of integrin-mediated cytoskeletal dynamics, such as phospho-p21-activated kinase (PAK1 Ser199-204/PAK2 Ser192-197; p=0.0005), phospho-LIM kinase (LIMK1 Thr508/LIMK2 Thr505; p=0.00001) and the adaptor protein CrkII Tyr221 (p=0.039). Moreover, CD49d pos CLL cells were characterized by a high correlation between proteins involved in integrin-mediated signal trasduction, including the focal-adhesion kinase (FAK Tyr576-Ser577), the tyrosine kinase Src Tyr527 and the adaptor protein CrkL Tyr207. Since PAK and LIMK represent key players in the modulation of the structure and the activity of actin cytoskeleton, we focused on these proteins for validation experiments. The constitutive pPAK and pLIMK overexpression in the CD49d pos CLL group was confirmed by western blot analysis comparing purified CLL cells from 3 CD49d pos versus 3 CD49d neg cases (mean fold increase >100 for both proteins). The results obtained suggest that integrin signalling is constitutively active in CD49d-expressing circulating CLL cells, pointing to a constitutive receptor engagement by CD49d ligands allegedly present in plasma. To test whether plasma constituents could modulate integrin-signaling proteins, CLL cells from 5 cases, expressing or not CD49d, were challenged with autologous plasma (1:3 dilution) for 30 seconds, 1 and 3 minutes. The presence of plasma induced a strong increase of PAK and LIMK phosphorylation intensities in CD49d pos CLL cells, starting at 30 seconds upon stimulation (mean fold increase >10 as compared to control for both proteins), and increasing after 1 and 3 minutes (mean fold increase >40 and >50 for both the proteins, respectively). Conversely, plasma stimulation did not induce pPAK and pLIMK expression modulation in CD49d neg CLL cells. Of note, pretreatment of CD49d-expressing CLL cells with the anti-CD49d HP1/2 blocking antibody, resulted in lower up-regulation of pPAK and pLIMK with an overall 60% inhibition for both the proteins (p=0.01), confirming the involvement of CD49d triggering in the observed activated signaling. Given the above results, we investigated plasma from 24 CLL patients, expressing CD49d at different levels, for the presence of the CD49d ligand fibronectin using an ELISA detection kit. All samples tested showed more than 1 mg/ml fibronectin concentration, without differences between CD49d-expressing and CD49d neg CLL cases. Altogether these results sustain the hypothesis of an active role of plasma/plasma components in the activation of CD49d-mediated integrin pathway, thus favoring the delivery of chemoresistance/pro-survival signals even in the context of circulating CLL cells. Our results may be of interest in the perspective of novel therapies (e.g. Bruton tyrosine kinase inhibitors) known to provoke a massive egress of neoplastic cells from tissue sites into the blood stream. Disclosures: No relevant conflicts of interest to declare.

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Novel Tyrosine Kinase Inhibitors Trigger Drug Sensitivity of Primary CLL Cells by Controlling Glucosylation of Ceramides

Blood ◽

10.1182/blood.v120.21.3905.3905 ◽

2012 ◽

Vol 120 (21) ◽

pp. 3905-3905

Author(s):

Janine Schwamb ◽

Valeska Feldhaus ◽

Michael Baumann ◽

Michaela Patz ◽

Susanne Brodesser ◽

...

Keyword(s):

Tyrosine Kinase ◽

Kinase Inhibitors ◽

Kinase Inhibitor ◽

Conflicts Of Interest ◽

Treatment Options ◽

Cell Receptor ◽

Lymphocytic Leukemia ◽

Published Data ◽

The Novel ◽

Apoptosis Resistance

Abstract Abstract 3905 Background: Apoptosis resistance of chronic lymphocytic leukemia (CLL) cells is mediated by several pro-survival stimuli. In particular, engagement of the B-cell receptor (BCR), CD40-CD40 ligand (CD40L) interaction or stimulation by interleukin-(IL)-4 were identified as major factors to regulate chemoresistance. Sphingolipids are known to be involved in several metabolic pathways involved in chemoresitance. Therefore, we focused on ceramide as pro-apoptotic molecule and its counterpart glucosylceramide, which rather contributes to proliferation and survival. Methods and Results: Applying liquid chromatography electrospray ionization tandem mass spectrometry, we revealed a significant decrease of pro-apoptotic ceramide in BCR/IL-4/CD40L-stimulated primary CLL cells compared to untreated controls (p=0.0258, p=0.0478, p=0.0114). Anti-apoptotic glucosylceramide levels were significantly increased after BCR cross-linking (p=0.0435) while other stimuli caused no relevant change in glucosylceramide expression. We identified BCR engagement to catalyze the crucial modification of ceramide to glucosylceramide via the enzyme UDP-glucose ceramide glucosyltransferase (UGCG) (p=0.0001). Besides specific UGCG inhibitors, we could show for the first time that IgM-mediated UGCG expression was significantly inhibited by the novel and highly effective PI3Kδ and BTK inhibitors CAL-101 and PCI-32765, which were able to revert IgM-induced apoptosis resistance of CLL cells. Recently published data revealed sphingolipids to be essential for mediation of apoptosis via mitochondria. Therefore, we chose ABT-737 – a well-known and also mitochondria-targeting drug – as candidate partner for PI3Kδ and BTK inhibition. When combining each tyrosine kinase inhibitor with ABT-737, a synergistic apoptotic effect could be documented, even under protection by BCR stimulation. Conclusion: In summary, we could demonstrate that sphingolipids are critically involved in CLL pathogenesis. UGCG could be identified as drugable target by the novel kinase inhibitors CAL-101 and PCI-32765 resulting in even synergistic apoptosis following additional application of ABT-737. Sphingolipids seem to offer further targets providing novel treatment options in CLL. C.M.W. and L.P.F. contributed equally to this work. Disclosures: No relevant conflicts of interest to declare.

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Serum IgM Activates BCR Signaling Pathways and Increases Survival of Chronic Lymphocytic Leukemia B Cells

Blood ◽

10.1182/blood.v126.23.4131.4131 ◽

2015 ◽

Vol 126 (23) ◽

pp. 4131-4131

Author(s):

Stefania Gobessi ◽

Binu K Sasi ◽

Luca Laurenti ◽

Dimitar G Efremov

Keyword(s):

B Cells ◽

Chronic Lymphocytic Leukemia ◽

Signaling Pathways ◽

Target Genes ◽

Conflicts Of Interest ◽

Direct Interaction ◽

Leukemic Cell ◽

Fold Increase ◽

Lymphocytic Leukemia ◽

Bcr Signaling

Abstract Serum IgM would be expected to bind chronic lymphocytic leukemia B cells through two different mechanisms. The first mechanism is via interactions between the immunoglobulin heavy chain CDR3 of the leukemic B cell receptors (BCRs) and internal epitopes located in the FR2 and FR3 regions of serum IgM molecules, analogous to the recently identified cell-autonomous BCR-BCR interaction. The latter interaction represents a general feature of human CLL BCRs and was recently shown to be positively selected during leukemia development in the Eμ-TCL1 transgenic murine model. The second mechanism is by binding of serum IgM to the recently identified Fc receptor for IgM (FcμR), which is overexpressed on CLL B cells. In the present study we investigated the consequences of the interaction between serum IgM and CLL cells. Incubation of CLL cells with Alexa488-conjugated human IgM resulted in strong cell surface labeling, confirming that IgM binds to CLL cells. Binding was substantially inhibited by preculture of CLL cells with Fcμ, suggesting that IgM interacts with CLL B cells primarily through the FcμR. To investigate whether IgM also binds to the leukemic BCRs, we analyzed activation of downstream BCR signaling pathways and expression of a well-defined set of BCR-target genes (Herishanu Y et al, Blood. 2011;117:563-74) in CLL cells cultured in the presence or absence of purified IgM. After three hours in culture with polyclonal or monoclonal human IgM, 5 of the 7 investigated BCR target genes (OAS3, RGS1, GFI1, CCND2 and KLF4) showed a 2- to 9-fold increase with respect to unstimulated CLL cells, whereas the remaining two genes (EGR1 and EGR2) were not induced. The induced BCR target genes were also upregulated to an equal or even greater extent by Fcμ, suggesting that these effects are primarily or exclusively caused by binding of IgM to the FcμR. Analysis of downstream signaling events, such as SYK and ERK phosphorylation, also showed similar induction by IgM and Fcμ. However, intracellular Ca2+ flux was induced to a substantially greater extent with IgM, suggesting that certain effects are mediated by a direct interaction between serum IgM and the leukemic cell BCRs. Since co-ligation of the FcμR was recently shown to enhance the survival of anti-IgM-stimulated murine B lymphocytes (Ouchida R et al, J Immunol. 2015;194:3096-101), we investigated the consequences of IgM binding on CLL cell survival. CLL cells from 18 patients were cultured with or without purified human IgM for 72 hours and then analyzed by Annexin V/PI staining. A modest but significant increase in the percentage of viable CLL cells was observed in the presence of IgM (percentage of viable CLL cells without IgM: 40.5±17.8; with IgM: 43.8±18.4; P =0.016), which was replicated in a smaller series of samples cultured with Fcμ (n=12, percentage of viable CLL cells without Fcμ: 41.1±17.8; with Fcμ: 49.5±15.6; P =0.019). Altogether, these data suggest that binding of serum IgM results in activation of prosurvival pathways in CLL cells and that this effect is most likely mediated by co-triggering the FcμR and BCR. Disclosures No relevant conflicts of interest to declare.

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NADPH Oxidases Are Important Regulators of Growth and Migration in Myeloid Neoplasms.

Blood ◽

10.1182/blood.v114.22.2190.2190 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2190-2190

Author(s):

Mamatha M. Reddy ◽

Margret Fernandes ◽

Ravi Salgia ◽

Ross L. Levine ◽

James D Griffin ◽

...

Keyword(s):

Cell Growth ◽

Tyrosine Kinase ◽

Myeloid Leukemia ◽

Conflicts Of Interest ◽

Myelogenous Leukemia ◽

Complex Iii ◽

Nadph Oxidases ◽

Myeloid Neoplasms ◽

Intracellular Ros ◽

And Migration

Abstract Abstract 2190 Poster Board II-167 Myeloid neoplasms are frequently associated with transforming tyrosine kinase oncogenes, including BCR-ABL in chronic myelogenous leukemia (CML), JAK2V617F in myeloproliferative disorders and FLT3.ITD in acute myeloid leukemia (AML). Cells transformed by tyrosine kinase oncogenes have been found to display increased levels of intracellular reactive oxygen species (ROS). Elevated ROS levels have been implicated in genomic instability through oxidative DNA damage, but also regulate cellular functions through redox-sensitive proteins. The majority of intracellular ROS in non-phagocytic cells are produced within the mitochondria. Nevertheless, recent studies indicate a role for NADPH oxidases (NOX) in transformation. There are five NOX genes (NOX1-5) and two genes encoding the related dual oxidases (DUOX1-2) in humans. We asked whether NOXs alter cell growth, migration and ROS production in hematologic malignancies transformed by tyrosine kinase oncogenes. Real-time PCR analysis in patient-derived cell lines KU812 (BCR-ABL), HEL (JAK2V617F) and Molm13 (FLT3.ITD), showed expression of NOX2, NOX4, and NOX5. Murine cells are readily transformed by BCR-ABL, JAK2V617F and FLT3.ITD, even though the gene for NOX5 is absent in mice. Therefore, we targeted NOX2 and NOX4 in KU812, HEL and Molm13 cells using lentiviral-based shRNAs. In addition, we also performed knockdown of p22phox, a common subunit that is required for stability and functioning of NOX1-4, thus also controlling for functional redundancy between the NOX genes. Interestingly, we found that p22phox knockdown reduced cell growth by 16.0-32.0% in KU812, 27.3-50.9% in HEL, and 31.3-54.2% in Molm13 cells (p<0.05, n=3). Similar to cell growth, a drastic reduction in spontaneous migration compared to control cells was observed in KU812 (74.3-87.6%, p<0.05) HEL (47.1-67.6%, p<0.05) and Molm13 (51.0-75.6%, p<0.05) cells with p22phox knockdown. Surprisingly, knockdown of NOX2, NOX4, or p22phox did not significantly reduce intracellular ROS levels. Further, mitochondrial electron transport chain inhibitors led to a decrease in intracellular ROS levels and oxygen consumption in KU812, HEL and Molm13 cells. Using patient samples, a similar decrease in ROS was observed in response to complex III inhibitors myxothiazol (CML: 57.2±9.4%; polycythemia vera: 36.2±0.4%; AML: 51.8±5.2%; p<0.05, n=4) and stigmatellin (CML: 62.5±7.8%; PV: 48.1±4.2%; AML: 53.9±5.5%; p<0.05, n=4). Consistent with the above data, KU812, HEL and Molm13 cells treated with their respective tyrosine kinase inhibitors, showed a reduction in MitoSOX Red fluorescence, an indication for reduced mitochondrial superoxide radicals (KU812: -38.9%; HEL: -31.3%; Molm13: -41.9%), further implicating mitochondria in the excess generation of intracellular ROS. Overall, these data suggest that expression of NOX proteins is required for optimal growth and migration of myeloid leukemia cells and therefore hints at novel targets for drug development that may aid traditional therapy. (supported in part by NIH/NCI grant 5R01CA134660-02) Disclosures: No relevant conflicts of interest to declare.

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Nucleophosmin-1 and Ribosome-Associated Components Are Constitutively Overexpressed in NOTCH1 Mutated IGHV Unmutated CLL

Blood ◽

10.1182/blood.v120.21.3880.3880 ◽

2012 ◽

Vol 120 (21) ◽

pp. 3880-3880

Author(s):

Michele Dal Bo ◽

Federico Pozzo ◽

Riccardo Bomben ◽

Antonella Zucchetto ◽

Erika Tissino ◽

...

Keyword(s):

Ribosomal Proteins ◽

Conflicts Of Interest ◽

Fold Increase ◽

Lymphocytic Leukemia ◽

Amplification Refractory Mutation System ◽

Qrt Pcr ◽

Mutational Status ◽

Frameshift Deletion ◽

Uptake Assay ◽

Notch1 Mutations

Abstract Abstract 3880 Background: activating mutations of NOTCH1 have been identified in about 10% of chronic lymphocytic leukemia (CLL) cases at diagnosis, with a higher frequency in unmutated IGHV (IGHV-UM) CLL, chemorefractory CLL and CLL in advanced disease phases. In CLL, all NOTCH1 mutations disrupt the C-terminal PEST domain and cause an accumulation of an active NOTCH1 isoform. Notably, about 80% of NOTCH1 mutations are represented by a CT frameshift deletion at nucleotides 7544–7545 (c.7544–7545delCT). Clinically, the presence of NOTCH1 mutations is an independent predictor of overall survival in CLL and identifies a subset of patients with particularly unfavourable prognosis (Rossi et al, Blood, 119, 521, 2012). Aim: to identify peculiar molecular and biological features of NOTCH1 mutated CLL in the context of IGHV-UM CLL. Methods: the presence of the c.7544–7545delCT NOTCH1 frameshift deletion was investigated by an ad-hoc amplification refractory mutation system (ARMS) PCR set up to obtain an amplicon specific for the NOTCH1 mutated form and a second amplicon as control. The percentage of NOTCH1 DNA in the context of the CLL clone was determined by quantitative real-time PCR (QRT-PCR), calculating the ratio between the amount of the specific NOTCH1 mutated amplicon and the amount of the control amplicon, the latter representing the total amount of NOTCH1 DNA irrespective of its mutational status. Gene expression profile (GEP) was performed by a one-color labeling strategy using the 4×44K Agilent platform. The differential expression of specific genes/proteins was validated by QRT-PCR, western blotting and immunohistochemistry. A BrdU uptake assay was used to evaluate proliferation of CLL cells by CpG/IL2 stimulation. Results: in a cohort of 380 IGHV-UM CLL, the c.7544–7545delCT NOTCH1 mutation was found in 83/380 (21.8%) cases. QRT-PCR revealed a percentage of NOTCH1 mutated DNA ranging from 1 to 37%. CLL cases carrying the c.7544–7545delCT NOTCH1 mutation (NOTCH1-Mut) showed higher NOTCH1 protein expression than CLL cases lacking NOTCH1-Mut employing monoclonal antibodies either recognizing the trans-membrane (mean fold increase=3) or the intra-citoplasmic (mean fold increase=2.1) NOTCH1 domain. A GEP comparing RNA from purified CLL samples of 5 NOTCH1-Mut CLL and 5 CLL lacking NOTCH1-Mut was performed, selecting the 5 NOTCH1-Mut cases among those with the higher percentages of NOTCH1 mutated DNA (percentages of NOTCH1 mutated DNA ranging from 15 to 37%). This approach selected the nucleophosmin 1 gene (NPM1) and genes codifying for several ribosomal proteins (RPS6, RPS10, RPS17, RPS28, RPSA, RPL7A, RPL18) as significantly up regulated in NOTCH1-Mut CLL cases. A higher expression of the above mentioned genes in NOTCH1-Mut CLL was validated in a wider series of 34 cases (18 NOTCH1-Mut cases; NPM1, p=0.03; RPS6, p=0.045; RPS10, p=0.048; RPS17, p=0.048; RPS28, p=0.049; RPSA, p=0.048; RPL7A, p=0.039; RPL18, p=0.041, respectively). Western blot analysis in 8 cases (4 NOTCH1-Mut cases) confirmed a higher NPM1 expression in NOTCH1-Mut cases (range of fold increase from 1.6 to 5.2) also at protein level. Consistently, lymph nodes preparations from NOTCH1-Mut CLL cases revealed a strong NPM1 staining both in nucleoli and cytoplasms. Finally, when stimulated in-vitro with the CpG/IL2 combination, NOTCH1-mut IGHV-UM CLL cells proliferated, as detected by a BrdU uptake assay (>10 fold increase over control), and up-regulated NPM1 both at transcript (mean fold increase=2.02 after 18 hours of CpG exposure, p=0.001) and protein (fold increase of 1.34 after 6 hours of CpG exposure) levels. Conclusion: NPM1 was identified as constitutively overexpressed in NOTCH1-Mut IGHV-UM CLL together with several ribosome-associated components. These findings are suggestive for an increased activity of the ribosomal machinery in NOTCH1-Mut IGHV-UM CLL as part of the molecular processes leading to control of CLL cell growth and survival in this clinically unfavourable disease subset. Disclosures: No relevant conflicts of interest to declare.

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The Combination Of 2-DG and Metformin Inhibits The mTORC1 Pathway and Suppresses Aggressive B Cell Lymphoma Growth and Survival

Blood ◽

10.1182/blood.v122.21.1665.1665 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1665-1665 ◽

Cited By ~ 2

Author(s):

Chengfeng Bi ◽

Kai Fu ◽

Chunsun Jiang ◽

Xin Huang ◽

Wing Chung Chan ◽

...

Keyword(s):

Cell Growth ◽

B Cell ◽

Conflicts Of Interest ◽

Cell Lymphoma ◽

Combined Treatment ◽

Control Cell ◽

B Cell Lymphoma ◽

Protein Translation ◽

Growth And Survival ◽

Aggressive B Cell Lymphoma

Abstract mTORC1 (mammalian target of rapamycin complex 1) plays a central role in integrating nutrient and growth factor inputs to control cell growth in all eukaryotes and is commonly deregulated in human cancers. Inhibition of mTORC1 is a promising strategy in lymphoma therapy. However, only a few drugs, such as rapamycin and its analogs (rapalogs), have been approved for treatment in a limited number of cancer types, due to their incomplete and nonspecific inhibition of mTORC1 as well as their limited effects toward the 4EBP1 pathway. 4EBP1 pathway regulates protein translation which is considered to be crucial in cancer cell survival and proliferation. In this study, we used the glycolysis inhibitor 2-deoxyglucose (2-DG) together with the mitochondrial respiratory inhibitor metformin to treat aggressive B cell lymphoma cells in vitro and in vivo. We found that the combined treatment inhibited mTORC1 and its major downstream targets, including 4EBP1. As a result, combined treatment significantly inhibited tumor cell growth and survival by the inhibition of 5’ cap-dependent translation involving lymphoma associated oncogenes such as MCL-1, BCL-XL and Cyclin D1. Moreover, the combination of 2-DG and metformin suppressed tumor growth in B cell lymphoma xenograft mouse models. Although the combined treatment dramatically decreased cellular ATP levels, mTORC1 inhibition was independent of AMPK activity but instead resulted from inhibitory effects on Rag-GTPases, which are upstream activators of mTORC1. Given that both 2-DG and metformin have been used in clinical diagnosis or treatment for decades, the combination of the two drugs hold promise as a new strategy to treat aggressive B cell lymphoma. Disclosures: No relevant conflicts of interest to declare.

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FLT3 TKI Are Differentially Affected By Both Plasma Binding and Stromal Survival Factors

Blood ◽

10.1182/blood.v128.22.1570.1570 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1570-1570

Author(s):

Young J David ◽

Hayley S Ma ◽

Donald Small

Keyword(s):

Clinical Trials ◽

Tyrosine Kinase ◽

Human Plasma ◽

Conflicts Of Interest ◽

Fold Increase ◽

Plasma Binding ◽

Independent Manner ◽

Survival Factors ◽

Flt3 Inhibitors ◽

The Impact

Abstract The FMS-like tyrosine kinase 3 (FLT3) is the most frequently mutated gene in acute myeloid leukemia (AML). As such it represents an attractive target for tyrosine kinase inhibitors (TKI) for the treatment of AML, a number of which have entered clinical trials. However, to date, these trials have demonstrated limited clinical efficacy. Evidence indicates that failure to achieved adequate inhibitory activity against FLT3, at least partially as a result of plasma protein binding, as well as survival factors provided by stroma, are among the reasons for the limited efficacy of FLT3 TKI. In this study we have characterized the effects of plasma binding and stromal survival factors on TTT-3002, a novel TKI that has demonstrated potent inhibition of the internal tandem duplication (FLT3-ITD) as well as a large panel of clinically described kinase mutants (FLT3-KD), and compared it to the effects these same conditions have on other FLT3 TKI. Using plasma from pediatric/young adult patients of a variety of ages (n = 24, median 7.2 years, 4.1-36.9 years), the impact of human plasma binding on the activity of TTT-3002 against FLT3-ITD dependent proliferation. 50% human plasma results in a moderate decrease in activity of TTT-3002 (IC50 45.2 nM in human plasma, 2.4 nM in media with 10% FCS). This is a markedly smaller shift than several TKIs currently or previously in clinical trials including sorafenib, CEP-701, PKC-412, and AC-220 (50 to >500-fold increase in IC50). The data demonstrate a clear linear relationship between plasma alpha-1 acidglycoprotein (AGP), an acute-phase reactant, and fold-increase in IC50 of TTT-3002 (1.7 fold-increase per 10 mg/dL AGP). The data also demonstrate a wide variability in patient plasma AGP concentration that is dependent upon age, but also appears to be significantly impacted by position in treatment (median 117 mg/dL, 58-354 mg/dL). Plasma AGP strongly inhibits several FLT3 inhibitors including CEP-701 and PKC-412 (>100-fold increase in IC50); whereas other FLT3 inhibitors such as sorafenib and AC-220 are inhibited by human plasma in an AGP-independent manner. These data indicate the presence of several human plasma proteins whose binding leads to drug inhibition that TTT-3002 is nonetheless resistant to, in comparison to other clinically relevant FLT3 inhibitors. The identification of these proteins is ongoing. Using primary human bone marrow stromal cell co-culture with two FLT3-ITD cell lines increases the IC50 of TTT-3002 but to a lower extent than that of AC-220 (MV4-11: 1.1-1.9 fold versus 2.3-2.7 fold, MOLM-14: 2.2-2.3 fold versus 3.5-3.6 fold). The data are consistent across stromal cells cultured from several different pediatric patients (n = 5). These data suggest that TTT-3002 may be more resistant to some of the inhibitory effects that have led to disappointing results with other similar FLT3-targeted TKIs. As such, TTT-3002 represents an appealing candidate for clinical studies. Disclosures No relevant conflicts of interest to declare.

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TLR-9 and IL-15-Driven Clonal Expansion of B-CLL Cells

Blood ◽

10.1182/blood.v124.21.1937.1937 ◽

2014 ◽

Vol 124 (21) ◽

pp. 1937-1937

Author(s):

Patricia K. A. Mongini ◽

Rashmi Gupta ◽

Charles C. Chu ◽

Joanna Stein ◽

Tatjana Stankovic ◽

...

Keyword(s):

Bone Marrow ◽

Cell Growth ◽

Lymph Nodes ◽

Conflicts Of Interest ◽

Leukemic Cell ◽

Clonal Expansion ◽

Lymphocytic Leukemia ◽

Clinical Progression

Abstract Clinical progression of B cell chronic lymphocytic leukemia (B-CLL) is linked to clonal growth within pseudo-follicles, typically within lymph nodes, bone marrow and spleen, and occasionally lungs and skin. Both the clone’s antigen receptor and stromal milieu appear to influence its growth rate. An involvement of TLR signals seems probable based on atypically elevated TLR-9 expression within B-CLL cells and the likelihood that the specificity of B-CLL antigen receptors (BCR) facilitates the internalization of molecules from apoptotic cells and/or microbes that are physically linked to CpG DNA. Nevertheless, recent findings that a large subset of B-CLL undergoes in vitro apoptosis upon stimulation with CpG-rich oligodeoxynucleotides (ODN) raised questions about a central role for TLR-9 signaling. Using a CFSE-based model for examining in vitro B-CLL clonal expansion/viability and a cohort consisting of 19 IGHV mutated (M) and 19 unmutated (U) B-CLL, we report that TLR-9 signaling is uniformly stimulatory when accompanied by signals from IL-15. Importantly, this cytokine is known to be constitutively produced by stromal cells in normal bone marrow, lymph nodes, and spleen and in a constitutive/inducible manner within skin and lungs. We show that B-CLL display reproducible inter-clonal differences in the number of division cycles attained and/or lymphoblast survival that were not linked to IGHV mutation status, but were statistically linked to whether the patient leukemic population contained subclones with trisomy-12 (p=0.0003) or contained subclones with both an ATM anomaly (11q22 del and/or ATM mutation) and 13q14 del (p=0.009). When all B-CLL clones were assessed, in vitro high-division or high-viability status in response to ODN + IL-15 was not statistically linked to clinical progression as determined by time to first treatment (TFT). Nonetheless, in vitro high-division status showed a statistically-significant direct linkage to patient survival (OS) (p=0.019 for OS within B-CLL manifesting > 50% cells with > 2 divisions versus B-CLL with < 50% cells with > 2 divisions). Subdivision of the total cohort into U-CLL and M-CLL subsets revealed that the link of high division status with overall survival is most characteristic of U-CLL. Immunohistological evidence of IL-15-producing cells within or proximal to Ki-67-positive pseudo-follicles in B-CLL-infiltrated spleen is consistent with a role for ODN + IL-15 signaling in promoting in vivo leukemic cell growth. Taken together, the findings from this study support the concept that in vivo B-CLL clonal expansion is dependent upon leukemic B-CLL homing to tissue sites where IL-15 is typically sequestered along with intrinsic properties of the B-CLL clone, e.g. cytogenetic anomalies within members of a B-CLL clone that heighten leukemic cell growth and/or survival and an expression of U-BCRs specific for apoptotic cell debris that increase the opportunity for TLR-9 signaling. Disclosures No relevant conflicts of interest to declare.

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NOTCH1 Mutations Are Associated with High CD49d Expression in Chronic Lymphocytic Leukemia

Blood ◽

10.1182/blood.v124.21.1978.1978 ◽

2014 ◽

Vol 124 (21) ◽

pp. 1978-1978

Author(s):

Antonella Zucchetto ◽

Chiara Caldana ◽

Federico Pozzo ◽

Silvia Rasi ◽

Carmela Ciardullo ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Conflicts Of Interest ◽

Statistical Significance ◽

Fold Increase ◽

Lymphocytic Leukemia ◽

Wild Type ◽

Apoptosis Resistance ◽

Mutation Burden ◽

Trisomy 12 ◽

Notch1 Mutations

Abstract Mutations of NOTCH1 have emerged as one of the most frequent somatic alterations in chronic lymphocytic leukemia (CLL), affecting up to 10–15% of patients. These mutations (~80% are 7544-7545delCT frameshift deletions) generate a truncated protein that accumulates in the cell and activates the downstream NOTCH1 signaling which is implicated in apoptosis resistance and increased survival of CLL cells. CD49d (α4 integrin chain) is one of the most relevant negative prognosticator in CLL, expressed by ~40% of CLL cases, and associated with aggressive/accelerated clinical courses, whose key role in CLL cell microenvironmental interactions has been thoroughly investigated. Literature data indicate that NOTCH1 has a role in activating the integrin signaling in several cell models. Given the higher CD49d expression characterizing trisomy 12 CLL, a CLL subset in which the NOTCH1 pathway is more often activated by NOTCH1 mutations, this study was aimed at investigating the contribution of NOTCH1 in the regulation of CD49d expression in CLL. The 7544-7545delCT NOTCH1 mutations were investigated by ARMS-PCR in 1027 CLL cases, all characterized for CD49d expression and for the cytogenetic profiles by FISH. NOTCH1 mutated cases were 158/1027 (15%), with a higher prevalence (36.7% of NOTCH1 mutated cases) in the trisomy 12 cytogenetic group. Analysis of CD49d expression highlighted a very strong association between the presence of NOTCH1 mutations and CD49d expression (p<0.0001). In particular, high CD49d expression (>30% of positive cells) was found in 102/158 (64.5%) NOTCH1 mutated cases as compared to 285/869 (32.8%) NOTCH1 wild-type cases. Of note, excluding trisomy 12 CLL, again NOTCH1 mutated CLL (100/865, 11.6%) displayed a significantly higher frequency of CD49d+ cases (52%) as compared to NOTCH1 wild-type CLL (25.7%) (p<0.0001). We next analyzed the percentage of mutated NOTCH1 DNA in the context of the CLL clone by a quantitative real-time PCR (QRT-PCR) approach set up by us to quantify the delCT NOTCH1 mutation. Using the 10% cut-off value to discriminate between cases with high (high NOTCH1) and low (low NOTCH1) mutation burden, 90/138 (67.7%) and 43/138 (32.3%) CLL cases were classified as low NOTCH1 and high NOTCH1, respectively. A higher prevalence of CD49d+ cases was found in the high NOTCH1 group (79%) as compared to the low NOTCH1 group (58%) (p=0.03). Moreover, a significant association between CD49d expression and a high NOTCH1 mutation burden was observed also excluding trisomy 12 CLL, with 69% of CD49d+ cases in the high NOTCH1 group, compared to 41% of CD49d+cases in the low NOTCH1 group (p=0.03). The association between NOTCH1 mutations and CD49d expression was next confirmed by next-generation sequencing results using the flow cytometrically sorted (>99% purity) CD49d- and CD49d+ components from 8 CLL cases characterized by both CD49d bimodal expression, and the presence of 7544-7545delCT NOTCH1 mutations at the subclonal level. In 7/8 cases, the CD49d+ component showed a higher NOTCH1 mutation burden compared to the CD49d- component, this difference reaching statistical significance in 4/7 cases. Of note, a similar clustering of mutations could not be observed in the CD49d- and CD49d+ components of other CLL cases characterized by bimodal CD49d expression and subclonal mutations of SF3B1 (n=1), BIRC3 (n=2) or TP53 (n=2). To verify whether NOTCH1 accumulation, as occurring in NOTCH1 mutated CLL, may influence CD49d expression, MEC-1 cells were transfected with a vector containing either a NOTCH1 intracellular domain (NICD) with 7544-7545delCT or a NICD carrying a missense mutation (c.5304G>A) generating a stop codon at the beginning of the sequence, as control. The higher levels of both NOTCH1 transcript (fold increase over control=2.2) and protein (fold increase over control=1.3) characterizing mutated-NICD MEC-1 cells, was paralleled by higher levels of CD49d expression (mean fluorescence intensity=23.300 versus 12.400) in these cells. Altogether our data demonstrate a direct correlation between NOTCH1 mutations and CD49d expression also outside the trisomy 12 CLL group, and suggest that accumulation of NOTCH1 may be directly or indirectly responsible for the up-regulation of CD49d expression. Disclosures No relevant conflicts of interest to declare.

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Signaling by Target of Rapamycin Proteins in Cell Growth Control

Microbiology and Molecular Biology Reviews ◽

10.1128/mmbr.69.1.79-100.2005 ◽

2005 ◽

Vol 69 (1) ◽

pp. 79-100 ◽

Cited By ~ 224

Author(s):

Ken Inoki ◽

Hongjiao Ouyang ◽

Yong Li ◽

Kun-Liang Guan

Keyword(s):

Cell Growth ◽

Molecular Mechanism ◽

Translational Control ◽

Mammalian Cells ◽

Control Cell ◽

Small Gtpase ◽

Initiation Factor ◽

Target Of Rapamycin ◽

Tor Signaling ◽

Ribosomal S6 Kinase

SUMMARY Target of rapamycin (TOR) proteins are members of the phosphatidylinositol kinase-related kinase (PIKK) family and are highly conserved from yeast to mammals. TOR proteins integrate signals from growth factors, nutrients, stress, and cellular energy levels to control cell growth. The ribosomal S6 kinase 1 (S6K) and eukaryotic initiation factor 4E binding protein 1(4EBP1) are two cellular targets of TOR kinase activity and are known to mediate TOR function in translational control in mammalian cells. However, the precise molecular mechanism of TOR regulation is not completely understood. One of the recent breakthrough studies in TOR signaling resulted in the identification of the tuberous sclerosis complex gene products, TSC1 and TSC2, as negative regulators for TOR signaling. Furthermore, the discovery that the small GTPase Rheb is a direct downstream target of TSC1-TSC2 and a positive regulator of the TOR function has significantly advanced our understanding of the molecular mechanism of TOR activation. Here we review the current understanding of the regulation of TOR signaling and discuss its function as a signaling nexus to control cell growth during normal development and tumorigenesis.

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Phosphorylation of Receptor Tyrosine Kinase ROR1 At Tyrosine 641, 646 and Serine 652 residues Might Be of Importance for the Survival of CLL Leukemic Cells

Blood ◽

10.1182/blood.v120.21.3885.3885 ◽

2012 ◽

Vol 120 (21) ◽

pp. 3885-3885

Author(s):

Mohammad Hojjat-Farsangi ◽

Amir Hossein Daneshmanesh ◽

Martin Norin ◽

Åsa Sandin ◽

Abdul Salam Khan ◽

...

Keyword(s):

Amino Acid ◽

Tyrosine Kinase ◽

Western Blot ◽

Tyrosine Kinases ◽

Conflicts Of Interest ◽

Annexin V ◽

Kinase Domain ◽

Lymphocytic Leukemia ◽

Leukemic Cells ◽

Tyrosine Kinase Domain

Abstract Abstract 3885 Background: Receptor tyrosine kinases (RTK) play crucial roles for different normal cellular processes as cell proliferation/differentiation, apoptosis and survival, as well as for the malignant phenotype of many types of cancer. ROR1, as a member of twenty RTKs families, has important functions during normal embryogenesis. ROR1 has shown to be overexpressed in chronic lymphocytic leukemia (CLL), mantle cell lymphoma and other hematological malignancies, as well as in solid tumors. ROR1 inhibition in CLL cells and cell lines with high expression of ROR1 induced specific apoptosis of the cells. In this study, we investigated the effects of an anti-ROR1 mAb against the extracellular CRD domain for specific dephosphorylation at the tyrosine kinase domain of ROR1 in CLL cells. The CRD domain contains a frizzle receptor, which is considered to be the ligand-binding region for e.g. Wnt5a. Wnt5a has been suggested to stimulate growth of CLL cells. Aims: To investigate phosphorylation of tyrosine and serine residues, within the tyrosine kinase domain of ROR1, of importance for survival of CLL cells. Methods: Bioinformatic analysis of the ROR1 structure revealed that three amino acid residues in the tyrosine kinase domain might be critically phosphorylated. Based on this prediction, a 19 amino acid long peptide, phosphorylated at two tyrosine (tyrosine 641, 646) and one serine (serine 652) residues was designed and used for immunization of rabbits. An anti-phospho-ROR1 (pROR1) polyclonal antibody (pAb) with high titers of anti-pROR1 pAb was purified, using phospho-peptide affinity chromatography. The specificity of anti-pROR1 pAb was determined by ELISA, immunoprecipitation (IP) and western blot experiments. An anti-ROR1 mAb (IgG) (CRD 1D8 clone) was used to analyse the effects on ROR1 phosphorylation in CLL cells at tyrosine 641, 646 and serine 652 residues preceding apoptosis. ROR1 phosphorylation was investigated by western blot and IP of ROR1 probed with anti-pROR1 pAb, from untreated and CLL cells treated with the anti-CRD 1D8 mAb. Quantitative intracellular staining of ROR1 by flowcytometry in time kinetics experiment after treatment with anti-CRD 1D8 mAb was also used to check phosphorylation of ROR1. Annexin V/PI staining (flowcytometry), MTT assay, PARP and caspase 8 cleavage as well as MCL-1 protein (western blot) were used for detection of apoptosis. To investigate phosphorylation and localization of 64–130 kDa ROR1 isoforms in various compartments of CLL cells, lysates were prepared from the nucleus and cytoplasmic proteins of CLL cells. Results: Two tyrosine (641, 646) residues and one serine (652) residue of the tyrosine kinase domain were phosphorylated in CLL cells. As previously described (Mellstedt et al, Abstract No: 1771, 53th ASH annals meeting, 2011), the 64, 105 and 130 kDa ROR1 isoforms were shown to be constitutively phosphorylated at tyrosine and serine residues in CLL leukemic cells. Treatment of CLL cells with an anti-ROR1 mAb against the CRD domain induced rapid dephosphorylation of ROR1 at tyrosine 641, 646 and serine 652 residues within 20 min and gradually increased up to 4 hours. The phosphorylated 64 kDa ROR1 isoform was localized to the nucleus of CLL cells and probably represents an intracellular part of ROR1, while the ROR1 130 kDa isoform was presented both in cytoplasm and nucleus of CLL cells. Conclusion: Our data show that the ROR1 molecule is phosphorylated at tyrosine 641, 646 and serine 652 residues. The presence of 64 and 130 kDa ROR1 isoforms in the nucleus of CLL cells may suggest a role of these isoforms as transcription factors. Collectively, the data might suggest that phosphorylated ROR1 may be an important protein for the growth of CLL cells as well as an interesting structure to target in a therapeutic intervention. Disclosures: No relevant conflicts of interest to declare.

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