CHIR258, a Novel Multi-Targeted Tyrosine Kinase Inhibitor, for the Treatment of t(4;14) Multiple Myeloma.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 641-641 ◽  
Author(s):  
Suzanne Trudel ◽  
Zhi Hua Li ◽  
Ellen Wei ◽  
Marion Wiesmann ◽  
Katherine Rendahl ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Mouse Model ◽  
Tyrosine Kinase ◽  
Cell Lines ◽  
Small Molecule ◽  
Kinase Inhibitor ◽  
Wild Type ◽  
Myeloma Cells

Abstract The t(4;14) translocation that occurs uniquely in a subset (15%) of multiple myeloma (MM) patients results in the ectopic expression of the receptor tyrosine kinase, Fibroblast Growth Factor Receptor3 (FGFR3). Wild-type FGFR3 induces proliferative signals in myeloma cells and appears to be weakly transforming in a hematopoeitic mouse model. The subsequent acquisition of FGFR3 activating mutations in some MM is associated with disease progression and is strongly transforming in several experimental models. The clinical impact of t(4;14) translocations has been demonstrated in several retrospective studies each reporting a marked reduction in overall survival. We have previously shown that inhibition of activated FGFR3 causes morphologic differentiation followed by apoptosis of FGFR3 expressing MM cell lines, validating activated FGFR3 as a therapeutic target in t(4;14) MM and encouraging the clinical development of FGFR3 inhibitors for the treatment of these poor-prognosis patients. CHIR258 is a small molecule kinase inhibitor that targets Class III–V RTKs and inhibits FGFR3 with an IC50 of 5 nM in an in vitro kinase assay. Potent anti-tumor and anti-angiogenic activity has been demonstrated in vitro and in vivo. We employed the IL-6 dependent cell line, B9 that has been engineered to express wild-type FGFR3 or active mutants of FGFR3 (Y373C, K650E, G384D and 807C), to screen CHIR258 for activity against FGFR3. CHIR258 differentially inhibited FGF-mediated growth of B9 expressing wild-type and mutant receptors found in MM, with an IC50 of 25 nM and 80 nM respectively as determined by MTT proliferation assay. Growth of these cells could be rescued by IL-6 demonstrating selectivity of CHIR258 for FGFR3. We then confirmed the activity of CHIR258 against FGFR3 expressing myeloma cells. CHIR258 inhibited the viability of FGFR3 expressing KMS11 (Y373C), KMS18 (G384D) and OPM-2 (K650E) cell lines with an IC50 of 100 nM, 250 nM and 80 nM, respectively. Importantly, inhibition with CHIR258 was still observed in the presence of IL-6, a potent growth factors for MM cells. U266 cells, which lack FGFR3 expression, displayed minimal growth inhibition demonstrating that at effective concentrations, CHIR258 exhibits minimal nonspecific cytotoxicity on MM cells. Further characterization of this finding demonstrated that inhibition of cell growth corresponded to G0/G1 cell cycle arrest and dose-dependent inhibition of downstream ERK phosphorylation. In responsive cell lines, CHIR258 induced apoptosis via caspase 3. In vitro combination analysis of CHIR258 and dexamethasone applied simultaneously to KMS11 cells indicated a synergistic interaction. In vivo studies demonstrated that CHIR258 induced tumor regression and inhibited growth of FGFR3 tumors in a plasmacytoma xenograft mouse model. Finally, CHIR258 produced cytotoxic responses in 4/5 primary myeloma samples derived from patients harboring a t(4;14) translocation. These data indicate that the small molecule inhibitor, CHIR258 potently inhibits FGFR3 and has activity against human MM cells setting the stage for a Phase I clinical trial of this compound in t(4;14) myeloma.

Inhibiting the IGF-1 Receptor Tyrosine Kinase with Picropodophillin: An In Vitro and In Vivo Study in the 5T33MM Mouse Model.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 640-640
Author(s):  
Karin Vanderkerken ◽  
Eline Menu ◽  
Thomas Stromberg ◽  
Hendrik De Raeve ◽  
Kewal Asosingh ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Mouse Model ◽  
Tyrosine Kinase ◽  
Receptor Tyrosine Kinase ◽  
Microvessel Density ◽  
Kinase Inhibitor ◽  
High Dose

Abstract Multiple myeloma (MM) represents a B-cell malignancy, characterized by monoclonal proliferation of plasma cells in the bone marrow (BM) and is associated with osteolysis and angiogenesis. Insulin-like growth factor-1 (IGF-1), produced by the BM stromal cells, has been described as an important factor in the survival, proliferation and migration of MM cells. The latter process is involved in the homing of the MM cells to the BM. IGF-1 also induces VEGF secretion by the MM cells, thus stimulating angiogenesis in the BM. As IGF-1 is a pleiotropic factor in MM, therapeutic strategies targeting the IGF-1R may be effective as anti-tumor treatments. In this work we investigated the effect of an IGF-1 receptor tyrosine kinase inhibitor (picropodophyllin or PPP1) in the murine, syngeneic 5T33MM model of multiple myeloma. This mouse model is representative for the human disease and can combine in vitro and in vivo studies. We first investigated the effects of PPP on the MM cells in vitro. We and others have previously demonstrated that IGF-1 induced ERK activation, involved in VEGF secretion and proliferation. When the 5T33MM cells were preincubated with 1microM PPP, Western blot analysis demonstrated the blocking of this activation. Furthermore, when the 5T33MM cells were preincubated with PPP for 30 min, IGF-1 induced VEGF secretion and proliferation of the 5T33MM cells were completely blocked. Next, we used the tyrosine kinase inhibitor PPP in vivo. 5T33MM cells were injected intravenously in C57BLKaLwRij mice and the development of the disease was monitored by measuring the serum paraprotein concentration. Mice were either treated with a low (17mM, IP, twice a day) or a high dose of PPP (50mM, IP, twice a day) or with the vehicle (DMSO/oil 9/1) from the day of injection with 5T33MM onward. At week 3, vehicle controls showed signs of morbidity and were sacrificed. The presence of tumor was measured by assessing serum paraprotein concentrations and determining the proportion of idiotype positive cells in the BM by flow cytometry. Angiogenesis was assessed by measuring the microvessel density on CD31 stained paraffin sections. The tumor burden in the bone marrow in the PPP treated mice was 77% lower than in vehicle treated animals (p< 0,0001) and the serum paraprotein concentration was 90% lower (p< 0,0001). The microvessel density in the BM of the PPP treated group was reduced by 60% (p< 0,02). In a separate survival experiment the mice were either treated with the vehicle or with the high dose (50mM) of PPP, from the time of tumor injection. Kaplan-Meier analysis demonstrated a significant increase in survival after treatment with PPP when compared with vehicle (28 vs. 18 days, p<0,001). These data demonstrate that the IGF-1RTK inhibitor PPP possesses strong anti-tumor activity, as demonstrated both in vitro and in vivo in a syngeneic model of multiple myeloma, and may therefore be an effective therapeutic candidate for MM treatment.

PKC412 Is a Multi-Targeting Kinase Inhibitor with Activity Against Multiple Myeloma In Vitro and In Vivo.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 247-247 ◽  
Author(s):  
Joseph Negri ◽  
Nicholas Mitsiades ◽  
Qingwei Deng ◽  
Zhaoqin Wen ◽  
David C. Geer ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Tyrosine Kinase ◽  
Cell Lines ◽  
Kinase Inhibitor ◽  
De Novo ◽  
Alkylating Agents ◽  
Nod Mice ◽  
Protective Effects

Abstract Multiple myeloma (MM) remains an incurable neoplasia and exhibits high propensity for de novo/acquired refractoriness even to novel agents, e.g. thalidomide (Thal) or proteasome inhibitors. This may be due to complex and evolving patterns of molecular lesions potentially conferring hyperactive antiapoptotic signaling with high degree of redundancy upon inhibition of isolated targets within those pathways. We thus hypothesized that, for genetically complex neoplasias such as MM, patient outcome might improved by addition, in the therapeutic armamentarium, of agents that simultaneously inhibit multiple proliferative/antiapoptotic targets. Towards this effort of multi-targeted therapies, we studied the tyrosine kinase inhibitor PKC412 (midausporin, Novartis, Basel, Switzerland). Low-nM levels of PKC412 selectively inhibit tyrosine kinase receptors, such as FLT3. But higher PKC412 concentrations (in μM range), which presumably inhibit (at least partly) other kinases, are achieved in clinical trials without catastrophic toxicities. This suggests that PKC412 can safely suppress in vivo the activity of, not just FLT3, but a broader spectrum of kinases, some of which (individually or cooperatively) might be critical for survival/proliferation of MM cells. Indeed, in vitro kinase activity assays showed that clinically achievable (high nM or low μM) PKC412 concentrations inhibit by &gt;20% important kinases, including p70S6K, IKK-a and Akt,. Consistent with such multi-targeted effect, in vitro MTT colorimetric survival assays showed activity of PKC412 (at sub-μM levels) against panel of 40 MM cell lines and 10 primary tumor cells from MM patients (including cells resistant to Dex, alkylating agents, anthracyclines, Thal or its immunomodulatory derivatives, bortezomib, and/or Apo2L/TRAIL), and cell lines from hematologic neoplasias, e.g. B- and T-ALL, CML, various non-Hodgkin’s lymphoma subtypes, and solid tumors (e.g. breast, prostate, lung, thyroid, ovarian, renal Ca, retinoblastoma and sarcomas)). Mechanistic studies confirmed that PKC412 blocks key growth/survival pathways (e.g. PI-3K/Akt, IKK-α/NF-κB), coupled with by downstream effects on suppression of diverse inhibitors of apoptosis (e.g. FLIP, XIAP, cIAP-2, survivin). These molecular sequelae explain, at least partly, the ability of PKC412 to sensitize MM cells to other anti-MM agents (such as Dex, cytotoxic chemotherapy or proteasome inhibitor bortezomib) and overcome protective effects of cytokines (e.g. IL-6) or bone marrow stromal cells. Importantly, PKC412 significantly prolonged the overall survival (p&lt;0.03, Kaplan-Meier analysis) of SCID/NOD mice in a clinically relevant model of diffuse MM bones lesions. These studies provide basis for clinical studies of PKC412 in MM and indicate that kinase inhibitors selectively blocking specific targets at low drug levels, may also have potent anti-tumor activities related to inhibition of multiple other, less specific, nonetheless important targets, thus allowing for anti-tumor activity in a much broader spectrum of tumor types than previously appreciated.

Trolox Improves the Sensitivity of Multiple Myeloma to Arsenic Trioxide in Vitro and In Vivo.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4920-4920
Author(s):  
Michael Sebag ◽  
Xian-Fang Huang ◽  
Nicolas Garnier ◽  
Wilson H. Miller ◽  
Koren Mann
Keyword(s):  
Oxidative Stress ◽  
Multiple Myeloma ◽  
Cell Growth ◽  
Mouse Model ◽  
Arsenic Trioxide ◽  
Cell Lines ◽  
Malignant Cells ◽  
Myeloma Cells

Abstract Abstract 4920 Arsenic trioxide (ATO) induces apoptosis and promotes differentiation of acute promyelocytic leukemia (APL) cells, but has less activity in other types of cancers. One factor that may impede ATO success outside of APL is its toxicity profile, which limits in vivo concentrations and therefore, therapeutic benefit. We have reported that trolox, an analogue of alpha tocopherol, can augment ATO sensitivity in a variety of malignant cells, while protecting non-malignant cells from ATO toxicity. In this current study, we have focused on Multiple Myeloma (MM), a plasma cell malignancy that often shows resistance to apoptosis, drug inhibition and remains incurable despite tremendous recent advances. Although ATO has activity against MM cells in vitro, clinical trials of ATO, given as a solo agent, in MM have shown limited promise. To see if the addition of trolox could augment ATO toxicity, a panel of human myeloma cell lines (HMCLs, n=9) representing the genetic diversity seen in this disease, were treated with increasing concentration of ATO with and without 100uM trolox. Cell growth was assessed by MTT viability assays and virtually all cell lines were sensitive to varying doses of ATO. Four cell lines (U266, KMS11, MM1R, MM1S) showed profound inhibition of cell growth with very low concentrations of ATO (<1uM). Trolox (100uM) alone had no effect on cell growth, but in concert with ATO further decreased cell growth by up to 50% as compared to the same dose of ATO alone in virtually all cell lines. To further elucidate the mechanism of growth inhibition, annexin V assays were performed by flow cytometry to measure apoptosis. In all cell lines (n=9), a clear increase in the apoptotic fraction was noted when trolox was added to varying doses of arsenic. To test whether oxidative stress plays a role in ATO-mediated apoptosis of myeloma cells, we looked at the induction of a stress response protein (HO-1), a marker of oxidative stress induced by ATO. Western blot analysis revealed that in all myeloma cells tested, HO-1 was dramatically and quickly induced by ATO and further induced by the addition of trolox, indicating a pro-oxidant activity of trolox in the malignant cells. While the mechanism of trolox enhancement of ATO function remains largely unknown, intracellular concentrations of ATO in MM cells, as measured by inductively coupled plasma mass spectrometry, suggest that trolox does not work by augmenting ATO import or intracellular accumulation. To test the efficacy of ATO with trolox in vivo, we used a novel transgenic mouse model of MM that has been shown to faithfully mimic the human disease and its response to treatment (Chesi et al, Cancer Cell 2008 Feb;13(2):167-80). We first treated MM afflicted mice with a low dose of ATO (5.0mg/kg) and Trolox (50mg/kg) to assess for toxicity and tolerability. This dose was well tolerated in all mice when given for 10 days with no obvious toxic effects. Serum protein electrophoresis performed at the end of the 10 day treatment period revealed that even at this low starting dose, one of three mice showed a 30% reduction in its paraprotein peak, while the others remained stable. Further studies with higher ATO concentrations in the same mouse model are underway. In conclusion, these data support the role of ATO plus Trolox, as a promising anti-myeloma therapy. Disclosures No relevant conflicts of interest to declare.

Mi-63, a Small Molecule Inhibitor of MDM2-p53 Interaction, Has Significant In Vitro Activity in Multiple Myeloma.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3464-3464
Author(s):  
Shaji Kumar ◽  
Michael Timm ◽  
Michael P. Kline ◽  
Jessica L. Haug ◽  
Teresa K. Kimlinger ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Death ◽  
Cell Lines ◽  
Small Molecule ◽  
Myeloma Cell ◽  
Small Molecule Inhibitor ◽  
Wild Type ◽  
Myeloma Cells ◽  
Molecule Inhibitor

Abstract Background: Multiple myeloma (MM) is a plasma cell proliferative disorder that results in considerable morbidity and mortality. As it is incurable with the current therapeutic approaches, more effective therapies based on better understanding of the pathobiology of the disease are needed. In MM, malignant plasma cells are characterized by low proliferative and apoptotic rates compared to other malignancies. The tumor suppressor gene p53, responsible for induction of cellular apoptosis in response to genotoxic stimuli, is relatively intact in most cases of myeloma. However, p53 mutations or deletion can occur late in the course of disease. Here we evaluate a novel small molecule inhibitor of the interaction between p53 and its negative regulator, MDM2, in the setting of myeloma. Methods and Results: Mi-63 was cytotoxic to several different myeloma cell lines with a median effect observed at approximately 2.5 μM in cell lines including MM1.S that express wild type p53 and between 10–15 μM in cells with mutated p53 as measured using an MTT cell viability assay. Additionally, Mi63 induced cytotoxicity in myeloma cell lines resistant to conventional agents such as Melphalan (LR50), Doxorubicin (Dox40) and Dexamethasone (MM1.R), indicating non-overlapping mechanisms. To evaluate the ability of the drug to induce cell death in the tumor microenvironment, MM cells were co-cultured with marrow stromal cells or in the presence of VEGF or IL-6, two cytokines known to be important for myeloma growth and survival. Mi63 was cytotoxic to myeloma cells under these conditions as well, at doses similar to those seen with myeloma cells alone. Mi63 was able to inhibit proliferation and induce apoptosis in myeloma cells in a dose- and time-dependent fashion, as demonstrated by flow cytometry using Annexin/PI staining as well as cell cycle studies. Treatment of myeloma cells with Mi63 was associated with early mitochondrial membrane depolarization, inversion of Bax/Bcl-2 ratio, and down regulation of Mcl-1, indicating induction of mitochondrial mechanisms of cell death. Mi63 was also cytotoxic to freshly isolated primary patient myeloma cells, inducing apoptosis in a dose-dependent manner. In the patient cells the drug appears to have a differential effect on the CD45 positive and negative cells. Conclusion: Mi-63 has significant activity in vitro in the setting of myeloma as demonstrated by its effect on myeloma cell lines and primary patient cells. It clearly induces apoptosis in myeloma cells, with higher activity seen in cells with wild type p53. Given the lack of p53 abnormalities in most of the patients with myeloma, this drug alone or in combination is likely to have significant clinical activity. Studies combining this with various DNA damaging drugs are in progress. These studies will eventually form the framework for future clinical studies.

In vitro and in vivo evaluations of the tyrosine kinase inhibitor NSC�680410 against human leukemia and glioblastoma cell lines

2002 ◽  
Vol 50 (6) ◽  
pp. 479-489 ◽  
Author(s):  
Ioannis A. Avramis ◽  
Garyfallia Christodoulopoulos ◽  
Atsushi Suzuki ◽  
Walter E. Laug ◽  
Ignacio Gonzalez-Gomez ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Kinase Inhibitor ◽  
Cell Lines ◽  
Kinase Inhibitor ◽  
Human Leukemia ◽  
Glioblastoma Cell ◽  
Glioblastoma Cell Lines

scholarly journals The Pim Kinase Inhibitor AZD1208 Enhances Apoptosis Induction By Clinically Active FLT3 Inhibitors in FLT3-ITD Acute Myeloid Leukemia Cells in Vitro and in Vivo through Synergistic Downregulation of Mcl-1 and of Bcl-xL

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3601-3601 ◽  
Author(s):  
Karthika Natarajan ◽  
Trevor J Mathias ◽  
Kshama A Doshi ◽  
Adriana E Tron ◽  
Manfred Kraus ◽  
...  
Keyword(s):  
Cell Lines ◽  
Myeloid Leukemia ◽  
Kinase Inhibitor ◽  
Wild Type ◽  
Protein Levels ◽  
Flt3 Inhibitors ◽  
Synergistic Induction ◽  
Induction Of Apoptosis

Abstract Internal tandem duplication (ITD) mutations of the receptor tyrosine kinase fms-like tyrosine kinase 3 (FLT3) are present in acute myeloid leukemia (AML) cells in 30% of cases and are associated with high relapse rate and short disease-free survival. FLT3 inhibitors have clinical activity, but their activity is limited and transient. New therapeutic approaches combining FLT3 inhibitors and inhibitors of downstream or parallel signaling pathways may increase depth and duration of responses. The Pim-1 serine/threonine kinase is transcriptionally upregulated by FLT3-ITD. We previously demonstrated that Pim-1 phosphorylates and stabilizes FLT3 and thereby promotes its signaling in a positive feedback loop. Pim kinase inhibitors are in clinical trials. Here we studied the effect of combinations of the Pim kinase inhibitor AZD1208 and clinically active FLT3 inhibitors on AML with FLT3-ITD in vitro and in vivo. Ba/F3-ITD cells, with FLT3-ITD, were grown in medium with the Pim kinase inhibitor AZD1208 at 1 μM and/or the FLT3 inhibitors quizartinib (Q), sorafenib (S) or crenolanib (C) at their IC50values of 1, 2.5 and 20 nM, respectively, and viable cells were measured at serial time points. While Q, S, C or AZD1208 treatments reduced cell numbers, compared to DMSO control, combined AZD1208 and Q, S or C treatments abrogated proliferation. Because FLT3-ITD cells remain responsive to FLT3 ligand (FLT3L) despite constitutive FLT3 activation and increased FLT3L levels following chemotherapy have been hypothesized to contribute to relapse, we repeated the proliferation experiments in the presence of 0, 1, 3 and 10 ng/ml FLT3L. FLT3L produced a concentration-dependent increase in proliferation and, while Q, S, C or AZD1208 treatments individually reduced cell numbers, combined AZD1208 and Q, S or C abrogated proliferation at all FLT3L concentrations tested, suggesting that these combinations overcome growth stimulation by FLT3L. To understand the anti-proliferative effect of combined Pim-1 and FLT3 inhibitors, we first studied cell cycle effects of AZD1208 and Q, S or C in Ba/F3-ITD cells and of AZD1208 and Q in the additional FLT3-ITD cell lines 32D-ITD, MV4-11 and MOLM14. We found a progressive increase in sub-G1 phase cells at 24, 48 and 72 hours, consistent with induction of apoptosis. Synergistic induction of apoptosis was confirmed by Annexin V/propidium iodide labeling of Ba/F3-ITD and 32D-ITD cells treated for 48 hours with AZD1208 combined with Q (p<0.0001), S (p<0.0001) or C (p<0.001), and of MV4-11 (p<0.0001) and MOLM14 (p<0.05) cells treated with AZD1208 combined with Q, in relation to each drug alone. Apoptosis was additionally confirmed by loss of mitochondrial membrane potential. Synergistic induction of apoptosis was not seen in Ba/F3-WT or 32D-WT cells, with wild-type FLT3, indicating a FLT3-ITD-specific effect. Synergistic (p<0.01) induction of apoptosis was seen in three FLT3-ITD AML patient samples treated in vitro with AZD1208 combined with Q. In an in vivo model, synergistic decrease in tumor volume was seen with combined AZD1208 and Q therapy in mice with subcutaneously implanted MV4-11 cells, with FLT3-ITD, but not with KG1a cells, with wild-type FLT3. Mechanistically, combined AZD1208 and Q treatment in vitro did not increase reactive oxygen species, compared to each drug alone, but increased both cleaved caspase 3 and cleaved poly (ADP-ribose) polymerase (PARP) levels, and caspase 3 cleavage was reduced by co-incubation with the pan-caspase inhibitor Z-VAD. Moreover, combined AZD1208 and Q treatment caused a synergistic decrease in expression of the anti-apoptotic Mcl-1 and of Bcl-xL proteins, but did not significantly alter Bim-1, p-Bad, Bad, Bax, Bak or Bcl-2, pro- and anti-apoptotic protein levels. Bcl-xL mRNA expression decreased along with protein levels, but Mcl-1 mRNA levels remain unchanged, indicating post-transcriptional down-regulation of Mcl-1 by the combination treatment. In summary, synergistic cytotoxicity of AZD1208 and clinically active FLT3 inhibitors was demonstrated in FLT3-ITD cell lines and patient samples in vitro and in cell lines in vivo, via caspase-mediated apoptosis, associated with a synergistic decrease in Mcl-1 and Bcl-xL expression. Our data suggest clinical promise for combination therapy with Pim kinase and FLT3 inhibitors in patients with AML with FLT3-ITD. Disclosures No relevant conflicts of interest to declare.

Mcl-1 Is Overexpressed in Multiple Myeloma and Correlates with Disease Severity.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1419-1419
Author(s):  
Soraya Wuilleme-Toumi ◽  
Nelly Robillard ◽  
Patricia Gomez-Bougie ◽  
Philippe Moreau ◽  
Steven Le Gouill ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Disease Severity ◽  
Cell Lines ◽  
Plasma Cells ◽  
Myeloma Cell ◽  
Lymphocytic Leukemia ◽  
Myeloma Cells

Abstract Multiple Myeloma (MM) is a fatal malignancy of B-cell origin characterized by the accumulation of plasma cells within the bone marrow. The expression of the pro-survival members of the Bcl-2 family has been shown to be a key process in the survival of myeloma cells. More particularly, Mcl-1 expression turned out to be critical for their survival. Indeed, knockdown of Mcl-1 by antisenses induces apoptosis in myeloma cells. Finally, Mcl-1 was found to be the only anti-apoptotic Bcl-2 family member which level of expression was modified by cytokine treatment of myeloma cells. For these reasons, we have evaluated the expression of Mcl-1 in vivo in normal, reactive and malignant plasma cells (PC) i.e., myeloma cells from 55 patients with MM and 20 human myeloma cell lines using flow cytometry. We show that Mcl-1 is overexpressed in MM in comparison with normal bone marrow PC. Forty-seven percent of patients with MM at diagnosis (p=.017) and 80% at relapse (p=.014 for comparison with diagnosis) overexpress Mcl-1. Of note, only myeloma cell lines but not reactive plasmocytoses have abnormal Mcl-1 expression, although both plasmocyte expansion entities share similar high proliferation rates (&gt;20%). Of interest, Bcl-2 as opposed to Mcl-1, does not discriminate malignant from normal PC. This shows that the overexpression of Mcl-1 is clearly related to malignancy rather than to proliferation. It will be important to know whether the overexpression of Mcl-1 is related to an abnormal response to cytokines like Interleukin-6 or to mutations of the promoter of the Mcl-1 gene as already described in B chronic lymphocytic leukemia. Finally, level of Mcl-1 expression is related to disease severity, the highest values being correlated with the shortest event-free survival (p=.01). In conclusion, Mcl-1 which has been shown to be essential for the survival of human myeloma cells in vitro is overexpressed in vivo in MM and correlates with disease severity. Mcl-1 represents a major therapeutical target in MM.

In Vitro and In Vivo Activity of the Src/Abl Kinase Inhibitor AP23464 and Analogs Against Activation Loop Mutants of KIT.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 793-793 ◽  
Author(s):  
Amie S. Corbin ◽  
Shadmehr Demehri ◽  
Ian J. Griswold ◽  
Chester A. Metcalf ◽  
William C. Shakespeare ◽  
...  
Keyword(s):  
Cell Lines ◽  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Myelogenous Leukemia ◽  
Activation Loop ◽  
Wild Type ◽  
Abl Kinase ◽  
Ic50 Values

Abstract Oncogenic mutations of the KIT receptor tyrosine kinase have been identified in several malignancies including gastrointestinal stromal tumors (GIST), systemic mastocytosis (SM), seminomas/dysgerminomas and acute myelogenous leukemia (AML). Mutations in the regulatory juxtamembrane domain are common in GIST, while mutations in the activation loop of the kinase (most commonly D816V) occur predominantly in SM and at low frequency in AML. Several ATP-competitive kinase inhibitors, including imatinib, are effective against juxtamembrane KIT mutants, however, the D816V mutant is largely resistant to inhibition. We analyzed the sensitivities of cell lines expressing wild type KIT, juxtamembrane mutant KIT (V560G) and activation loop mutant KIT (D816V,F,Y and murine D814Y) to a potent Src/Abl kinase inhibitor, AP23464, and analogs. IC50 values for inhibition of cellular KIT phosphorylation by AP23464 were 5–11 nM for activation loop mutants, 70 nM for the juxtamembrane mutant and 85 nM for wild type KIT. Consistent with this, IC50 values in cell proliferation assays were 3–20 nM for activation loop mutants and 100 nM for wild type KIT and the juxtmembrane mutant. In activation loop mutant-expressing cell lines, AP23464, at concentrations ≤50 nM, induced apoptosis, arrested the cell cycle in G0/G1 and down-regulated phosphorylation of Akt and STAT3, signaling pathways critical for the transforming capacity of mutant KIT. In contrast, 500 nM AP23464 was required to induce equivalent effects in wild-type KIT and juxtamembrane mutant-expressing cell lines. These data demonstrate that activation loop KIT mutants are considerably more sensitive to inhibition by AP23464 than wild type or juxtamembrane mutant KIT. Non-specific toxicity in parental cells occurred only at concentrations above 2 μM. Additionally, at concentrations below 100 nM, AP23464 did not inhibit formation of granulocyte/macrophage and erythrocyte colonies from normal bone marrow, suggesting that therapeutic drug levels would not impact normal hematopoiesis. We also examined in vivo target inhibition in a mouse model. Mice were subcutaneously injected with D814Y-expressing (D816V homologous) murine mastocytoma cells. Once tumors were established, compound was administered three-times daily by oral gavage. One hour post treatment we observed >90% inhibition of KIT phosphorylation in tumor tissue. Following a three-day treatment regimen, there was a statistically significant difference in tumor size compared to controls. Thus, AP23464 analogs effectively target D816-mutant KIT both in vitro and in vivo and inhibit activation loop KIT mutants more potently than the wild type protein. These data provide evidence that this class of kinase inhibitors may have therapeutic potential for D816V-expressing malignancies such as SM or AML.

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