Venetoclax Is Effective in Small-Cell Lung Cancers with High BCL-2 Expression.

Purpose: Small-cell lung cancer (SCLC) is an often-fatal neuroendocrine carcinoma usually presenting as extensive disease, carrying a 3% 5-year survival. Despite notable advances in SCLC genomics, new therapies remain elusive, largely due to a lack of druggable targets.Experimental Design: We used a high-throughput drug screen to identify a venetoclax-sensitive SCLC subpopulation and validated the findings with multiple patient-derived xenografts of SCLC.Results: Our drug screen consisting of a very large collection of cell lines demonstrated that venetoclax, an FDA-approved BCL-2 inhibitor, was found to be active in a substantial fraction of SCLC cell lines. Venetoclax induced BIM-dependent apoptosis in vitro and blocked tumor growth and induced tumor regressions in mice bearing high BCL-2-expressing SCLC tumors in vivo BCL-2 expression was a predictive biomarker for sensitivity in SCLC cell lines and was highly expressed in a subset of SCLC cell lines and tumors, suggesting that a substantial fraction of patients with SCLC could benefit from venetoclax. Mechanistically, we uncover a novel role for gene methylation that helped discriminate high BCL-2-expressing SCLCs.Conclusions: Altogether, our findings identify venetoclax as a promising new therapy for high BCL-2-expressing SCLCs. Clin Cancer Res; 24(2); 360-9. ©2017 AACR.

Combination with vorinostat overcomes ABT-263 (navitoclax) resistance of small cell lung cancer.

Small cell lung cancer (SCLC) is an aggressive tumor type with high mortality. One promising approach for SCLC treatment would be to utilize agents targeting molecular abnormalities regulating resistance to apoptosis. BH3 mimetic antagonists, such as ABT-737 and its orally available derivative ABT-263 (navitoclax) have been developed to block the function of pro-survival BCL-2 family members. The sensitivity of SCLC to these drugs varies over a broad range in vitro and in clinical trials. We have previously shown that the expression of Noxa, a BH3-only pro-apoptotic BCL-2 family protein, is a critical determinant of sensitivity to ABT-737. Thus, pharmacological up-regulation of Noxa could enhance cell death induced by the BH3 mimetics. We find that the combination of ABT-263 and a HDAC inhibitor, vorinostat, efficiently induces apoptosis in a variety of SCLC cell lines, including ABT-263 resistant cell lines. Cell death induced by combined treatment is Noxa- and/or BIM-dependent in some cell lines but in others appears to be mediated by down-regulation of BCL-XL and release of BAK from BCL-XL and MCL-1. These results suggest that combination of HDAC inhibitors and BCL-2 inhibitors could be an alternative and effective regimen for SCLC treatment.

Early FDG/PET scanning as a pharmacodynamic marker of anti-EGFR antibody activity in colorectal cancer.

Panitumumab is an anti-EGF receptor (EGFR) antibody approved for use in treatment of chemotherapy-refractory colorectal cancers lacking K-RAS mutations. Despite overall response rates approximating 10%, no marker predictive of clinical benefit has been identified. We describe a chemotherapy-refractory patient whose clinical condition necessitated rapid identification of an effective agent in whom we used (18)F-fluorodeoxyglucose-positron emission tomography (FDG-PET)/computed tomographic scanning 48 hours after an initial dose of panitumumab to document a pharmacodynamic response to the antibody. The initial 46% ± 2.7% drop in SUV(max) of four target lesions correlated with a partial response by Response Evaluation Criteria in Solid Tumors and a >90% drop in serum carcinoembryonic antigen at 8 weeks, indicating that an early decrease in FDG uptake may predict subsequent clinical benefit in response to anti-EGFR antibody therapy in colorectal cancer.

Actinomycin D decreases Mcl-1 expression and acts synergistically with ABT-737 against small cell lung cancer cell lines.

PURPOSE: ABT-737, which blocks the function of Bcl-2 and Bcl-X(L) but not Mcl-1, has shown single-agent activity in preclinical models of small cell lung cancer (SCLC). Elevated expression of Mcl-1 induces resistance to ABT-737 in SCLC. Based on the short half-life of Mcl-1 mRNA and protein, we hypothesized that the actinomycin D could reverse Mcl-1-induced resistance to ABT-737. EXPERIMENTAL DESIGN: The dose-response of multiple SCLC cell lines to actinomycin D in the absence and presence of ABT-737 was followed by the assessment of Bcl-2 family expression and poly ADP ribose polymerase cleavage by Western blot, viability by tetrazolium dye reduction and clonogenic assay, and cell cycle kinetics by flow cytometry. RESULTS: Actinomycin D decreased Mcl-1 expression and resulted in a cell line-dependent increase in Noxa expression. Clinically relevant concentrations of actinomycin D from 0.4 to 4 ng/mL showed single-agent activity across a panel of SCLC cell lines. When combined with low micromolar doses of ABT-737, near complete loss of viability was seen with synergistic combination indices of 0.5 to 0.7. Exposure to 4 ng/mL actinomycin was only required for the first 24 hours of the combined incubation, mimicking a clinically achievable area under the curve, but the presence of ABT-737 was required for an additional 48 hours to obtain maximal effect. CONCLUSIONS: Clinically relevant concentrations of actinomycin D act synergistically with ABT-737 to induce SCLC apoptosis, which can be at least partially attributed to the actinomycin D-induced decrease in Mcl-1 and increase in Noxa expression. Taken together, these data suggest the feasibility of combining actinomycin D with BH3-mimetic drugs in the clinical setting.

Alterations in the Noxa/Mcl-1 axis determine sensitivity of small cell lung cancer to the BH3 mimetic ABT-737.

To understand the molecular basis for variable sensitivity to the BH3 mimetic drug ABT-737, the abundance of Bcl-2 family members was assayed in a panel of small cell lung cancer cell lines whose sensitivity varied over a 2-log range. Elevated Noxa and Bcl-2 levels directly correlated with sensitivity to ABT-737, whereas Mcl-1 levels were similar in all cell lines tested regardless of sensitivity. Transgenically enforced expression of Noxa but not Bcl-2 resulted in increased sensitivity to ABT-737 in multiple cell lines. This increase was especially pronounced in the H209 cell line in which expression of Noxa resulted in a proportionate decline in Mcl-1 expression. Although overexpression of Noxa enhanced sensitivity of the H526 and H82 cell lines to ABT-737, it did not result in altered Mcl-1 levels. Similarly, small interfering RNA-mediated knockdown of Noxa expression in the H146 cell line, which increased resistance to ABT-737, did not result in altered Mcl-1 levels. Therefore, three of four cell lines studied failed to show Noxa-mediated regulation of Mcl-1 expression. However, despite failure to regulate Mcl-1 levels, Noxa blocked binding of Bim to Mcl-1 following its release from Bcl-2 by ABT-737. Finally, we observed that a 24-hour incubation of the H526 and WBA cell lines with ABT-737 resulted in increased Noxa expression, suggesting that Noxa may play a direct role in ABT-737-mediated apoptosis. These results indicate that Noxa expression is the critical determinant of ABT-737 sensitivity and loss of Noxa-mediated regulation of Mcl-1 expression may be an important feature of small cell lung cancer biology.

Selective inhibition of SCLC growth by the A12 anti-IGF-1R monoclonal antibody correlates with inhibition of Akt.

Activation of the insulin-like growth factor-1 receptor (IGF-1R) by IGF-1 and IGF-2 plays a prominent role in the growth and survival of small cell lung cancer (SCLC) by potently activating the PI3K-Akt signal transduction pathway, which is also an important factor in the resistance of SCLC to chemotherapy. A12 is a fully human monoclonal antibody directed against the human IGF-1R that does not cross-react with the insulin receptor. In this study we have utilized A12 to determine the effects of selective antibody-mediated blockade of the IGF-1R on SCLC cell lines. Incubation with A12 resulted in a dose-dependent inhibition of IGF-1-stimulated IGF-1R and Akt activity, with maximal inhibition of approximately 75% at a concentration of 10mug/ml in the H526 cell line. Growth of the H526 and H146 cell lines in serum was inhibited by a maximum of 50-70% in a dose-dependent fashion, which correlated well with the extent of Akt inhibition. However, growth of the H69 and WBA cell lines was unaffected by A12. Despite almost complete inhibition of IGF-1R phosphorylation by A12, Akt activity remained constitutively high in these cell lines. H526 transfectants expressing a constitutively active Akt allele also were resistant to A12. Treatment with A12 additively enhanced response to carboplatin in the H526 and H146 cell lines but had no effect on the H69 and WBA cell lines. Treatment of the H526 cell line with a combination of A12 and rapamycin was highly synergistic. These data suggest that growth inhibition and chemosensitization of SCLC by A12 is directly correlated with the ability to inhibit PI3K-Akt signaling, with those cell lines showing constitutive PI3K-Akt signaling displaying a high level of resistance to IGF-1R targeted therapy.

Selective compounds define Hsp90 as a major inhibitor of apoptosis in small-cell lung cancer.

The heat shock protein 90 (Hsp90) has a critical role in malignant transformation. Whereas its ability to maintain the functional conformations of mutant and aberrant oncoproteins is established, a transformation-specific regulation of the antiapoptotic phenotype by Hsp90 is poorly understood. By using selective compounds, we have discovered that small-cell lung carcinoma is a distinctive cellular system in which apoptosis is mainly regulated by Hsp90. Unlike the well-characterized antiapoptotic chaperone Hsp70, Hsp90 is not a general inhibitor of apoptosis, but it assumes this role in systems such as small-cell lung carcinoma, in which apoptosis is uniquely dependent on and effected through the intrinsic pathway, without involvement of caspase elements upstream of mitochondria or alternate pathways that are not apoptosome-channeled. These results provide important evidence for a transformation-specific interplay between chaperones in regulating apoptosis in malignant cells.

Imatinib inhibits c-Kit-induced hypoxia-inducible factor-1alpha activity and vascular endothelial growth factor expression in small cell lung cancer cells.

Vascular endothelial growth factor (VEGF) is one of the most important mediators of tumor angiogenesis. In addition to hypoxia, peptide growth factors are known to regulate VEGF expression but the effect of stem cell factor (SCF), the ligand for c-Kit, on VEGF expression has not been characterized. We therefore studied the effect of SCF-mediated c-Kit activation on VEGF expression by the H526 small cell lung cancer (SCLC) cell line. SCF treatment doubled VEGF mRNA expression and VEGF secretion in the absence of other exogenous growth factors, an effect efficiently blocked by imatinib. The increase in VEGF mRNA occurred within the first 2 hours of treatment and was not caused by alterations in mRNA stability. The phosphatidylinositol 3-kinase inhibitor LY294002 blocked the increase in VEGF mRNA, implicating c-Kit-mediated activation of phosphatidylinositol 3-kinase in the phenomenon. VEGF promoter-reporter transfections indicated that a SCF-mediated increase in VEGF promoter activity paralleled the increase in VEGF mRNA, documenting that SCF mediated its effects through enhanced VEGF transcription. Mutation of the core hypoxia-inducible factor (HIF)-1 binding element in the VEGF promoter significantly blunted SCF-responsiveness. SCF increased nuclear levels of the HIF-1alpha transcription factor, which correlated well with increased HIF-1alpha binding to a consensus hypoxia-responsive element. SCF-mediated effects on HIF-1alpha expression were additive with those produced by CoCl2, a hypoxia-mimetic agent. These data indicate that activation of c-Kit by SCF leads to a predominantly HIF-1alpha-mediated enhancement of VEGF expression and that inhibition of c-Kit signaling with imatinib could result in inhibition of tumor angiogenesis.

Inhibition of insulin/IGF-1 receptor signaling enhances bile acid toxicity in primary hepatocytes.

Modulation of ERBB and insulin-like growth factor 1 (IGF-1) receptor function is recognized as a potential mechanism to inhibit tumor growth. We and others have shown that inhibition of ERBB1 can enhance bile acid toxicity. Herein, we extend our analyses to examine the impact of insulin/IGF-1 receptor inhibition on primary hepatocyte survival when exposed to the secondary bile acid deoxycholic acid (DCA) and compare the impact inhibition of this receptor has on bile acid toxicity effects to that of ERBB1/MEK1/2 inhibition. The insulin/IGF-1 receptor inhibitor NVP-ADW742 at concentrations which inhibit both the insulin and IGF-1 receptors had a modest negative impact on hepatocyte viability, and strongly potentiated DCA-induced apoptotic cell death. Identical data were obtained expressing a dominant negative IGF-1 receptor in hepatocytes; a receptor which acts to inhibit both the IGF-1 receptor and the insulin receptor in trans. Inhibition of ERBB1 function using Iressa (gefitinib) or the tyrphostin AG1478 had more modest effects at enhancing DCA lethality than inhibition of the insulin/IGF-1 receptor function. In contrast, over-expression of a dominant negative ERBB1 protein had pleiotropic effects on multiple signaling pathways in an apparently non-specific manner. These findings suggest that novel therapeutic kinase inhibitors, targeted against growth factor receptors, have the potential to promote bile acid toxicity in hepatocyte when bile flow may be impaired.

The insulin-like growth factor-I receptor kinase inhibitor, NVP-ADW742, sensitizes small cell lung cancer cell lines to the effects of chemotherapy.

PURPOSE: Insulin-like growth factor-I (IGF-I) is a potent growth factor for small cell lung cancer (SCLC) in both the autocrine and endocrine context. It also inhibits chemotherapy-induced apoptosis through activation of the phosphatidylinositol 3-kinase (PI3K)-Akt pathway and we have previously shown that inhibition of this signaling pathway enhances sensitivity of SCLC cell lines to chemotherapy. The purpose of this study was to determine whether the novel IGF-I receptor (IGF-IR) kinase inhibitor, NVP-ADW742, sensitizes SCLC cell lines to etoposide and carboplatin, which are commonly used in the treatment of SCLC. EXPERIMENTAL DESIGN: Cell growth in the presence of various combinations of NVP-ADW742, imatinib (STI571; Gleevec/Glivec), and chemotherapeutic agents was monitored using a 3-(4,5 dimethylthiazol-2yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay and analyzed using the Chou-Talalay multiple-drug-effect equation. Induction of apoptosis was assessed using terminal deoxynucleotide transferase-mediated dUTP nick end labeling (TUNEL) and Western blot analysis of procaspase 3 and poly(ADP-ribose)polymerase cleavage. IGF-I-induced vascular endothelial cell growth factor expression was monitored by Northern blot and ELISA. RESULTS: NVP-ADW742 synergistically enhanced sensitivity of multiple SCLC cell lines to etoposide and carboplatin. Maximal enhancement occurred at concentrations of NVP-ADW742 that eliminated basal PI3K-Akt activity in individual cell lines. In the WBA cell line, in which the c-Kit receptor tyrosine kinase is partly responsible for basal PI3K-Akt activity, the combination of NVP-ADW742 and imatinib was superior to NVP-ADW742 alone in sensitizing the cells to etoposide. Enhancement of the sensitivity of SCLC cell lines to etoposide, as determined by MTT assay, correlated closely with sensitization to the induction of apoptosis as measured by TUNEL and caspase activation assays. Treatment with NVP-ADW742 also eliminated IGF-I-mediated expression of vascular endothelial cell growth factor, suggesting that in addition to enhancing sensitivity of SCLC to chemotherapy, this kinase inhibitor could potentially inhibit angiogenesis in vivo. CONCLUSIONS: Inhibition of IGF-IR signaling synergistically enhances the sensitivity of SCLC to etoposide and carboplatin. This enhancement in sensitivity to chemotherapy tightly correlates with inhibition of PI3K-Akt activation. Future SCLC clinical trials incorporating IGF-IR inhibitors alone or in combination with other kinase inhibitors should include assessment of PI3K-Akt activity as a pharmacodynamic end-point.

The multi-targeted kinase inhibitor SU5416 inhibits small cell lung cancer growth and angiogenesis, in part by blocking Kit-mediated VEGF expression.

SU5416 is a multi-targeted kinase inhibitor that potentially has the ability to directly block tumor growth by inhibiting Kit signaling, as well as blocking angiogenesis by inhibiting vascular endothelial growth factor receptor (VEGFR) signaling. Previous work has demonstrated that SU5416 efficiently blocks Kit-mediated growth of small cell lung cancer (SCLC) in vitro. To determine the drug's effect on in vivo growth of SCLC, we studied its activity, alone and in combination with carboplatin, in chemotherapy-resistant H526, and chemotherapy-sensitive H209 murine xenograft models. SU5416 efficiently inhibited Kit activity in vivo when administered on a twice-weekly schedule. When administered over a 3-week period to animals bearing established tumors, it inhibited growth by at least 70%. It was at least as effective as carboplatin in suppressing growth of H526 xenografts. However, the combination with carboplatin was not superior to the most active single agent in either xenograft model at the doses and schedule utilized. SU5416 clearly inhibited growth in part by inhibiting angiogenesis, with microvessel density dropping by approximately 50% in treated xenografts. In addition to the recognized mechanism of inhibition of VEGFR, we uncovered a novel mechanism of angiogenesis suppression by demonstrating reduced VEGF expression in SU5416-treated xenografts. In vitro, stem cell factor treatment of the H526 cell line enhanced expression of VEGF, which was efficiently blocked with SU5416. Thus, we have demonstrated that SU5416 can inhibit SCLC growth by directly inhibiting tumor cell proliferation and by inhibiting angiogenesis, in part by inhibiting Kit-mediated VEGF expression. These data suggest that kinase inhibitors that target both Kit and VEGFR could play an important role in the treatment of SCLC, as well as other malignancies that express Kit.

The insulin-like growth factor-I (IGF-I) receptor kinase inhibitor NVP-ADW742, in combination with STI571, delineates a spectrum of dependence of small cell lung cancer on IGF-I and stem cell factor signaling.

Stem cell factor (SCF)/Kit and insulin-like growth factor-I (IGF-I)/IGF-I receptor (IGF-IR) autocrine loops play a prominent role in the growth of small cell lung cancer (SCLC). Previous data suggested that IGF-I protects cells from apoptosis induced by STI571, an efficient inhibitor of Kit signal transduction, by activating the critical phosphatidylinositol 3-kinase-Akt pathway. To determine if inhibition of IGF-IR signaling would be therapeutically relevant in SCLC, the activity of a novel kinase inhibitor of IGF-IR, NVP-ADW742 (Novartis Pharma AG, Basel, Switzerland), was characterized. Pretreatment of the H526 cell line with NVP-ADW742 inhibited IGF-IR signaling and growth with IC(50) values between 0.1 and 0.4 micro M. SCF-mediated Kit phosphorylation and Akt activation were inhibited with IC(50) values in the 1-5 micro M range. However, NVP-ADW742 affected neither hepatocyte growth factor-mediated Akt activation nor activity of constitutively active Akt. The therapeutic potential of NVP-ADW742 was assessed by determining its effect on growth of several SCLC cell lines in serum. These studies clearly delineated two populations of cell lines as determined by differential sensitivity to NVP-ADW742. One population, which lacks active SCF/Kit autocrine loops, was inhibited with IC(50) values between 0.1 and 0.5 micro M. A second population, which has active SCF/Kit autocrine loops, was inhibited with IC(50) values in the 4-7 micro M range. When these cell lines were treated with a combination of STI571 and NVP-ADW742, no advantage was seen in the former group, whereas, in the latter group, a clearly synergistic response to the combination was seen when growth, apoptosis, or Akt activation was assessed. These data demonstrate that NVP-ADW742 is a potent and selective IGF-IR kinase inhibitor that can efficiently inhibit the growth of cells that are highly dependent on IGF-I signaling. However, for optimal growth inhibition of SCLC cells with an active SCF/Kit autocrine loop, a combination of a Kit inhibitor (STI571) and an IGF-IR inhibitor (NVP-ADW742) appears to be necessary. These observations suggest that, in tumors in which critical signal transduction pathways can be activated by alternative receptors, optimal therapy may require inhibition of multiple receptors.

Imatinib mesylate (STI571) for myeloid malignancies other than CML.

Imatinib mesylate, a small molecule tyrosine kinase inhibitor, has had a major impact on the treatment of Philadelphia chromosome positive chronic myelogenous leukemia. This review will explore its potential in the treatment of other myeloid neoplasms, based on its ability to inhibit Kit and PDGFR kinases in addition to Bcr-Abl. Imatinib's potential role in the treatment of Philadelphia chromosome negative chronic myelogenous leukemia, systemic mastocytosis with associated hematologic neoplasms, chronic myelomonocytic leukemia, specific subtypes of acute myelogenous leukemia, myelofibrosis/myeloid metaplasia, and polycythemia vera is discussed.

Flavopiridol potently induces small cell lung cancer apoptosis during S phase in a manner that involves early mitochondrial dysfunction.

PURPOSE: Accumulating evidence indicates that small cell lung cancer (SCLC) is defective in many of the regulatory mechanisms that control cell cycle progression. The purpose of this study was to determine the effects of flavopiridol, a pan-cyclin-dependent kinase inhibitor, on growth and apoptosis of SCLC cell lines. EXPERIMENTAL DESIGN: Cell growth was monitored using 3-(4,5dimethylthiazol-2yl)-2,5-diphenyl-tetrazolium bromide (MTT) and clonogenic assays. Induction of apoptosis was assessed using multiple assays, including flow cytometric determination of DNA content and mitochondrial membrane potential, terminal deoxynucleotide transferase-mediated dUTP nick end labeling (TUNEL), and Western blot analysis of procaspase 3 and poly(ADP-ribose) polymerase cleavage. RESULTS: Flavopiridol induced growth inhibition and cytotoxicity in multiple SCLC cell lines, with an IC(50) of 50-100 nM and an LD(50) of 150-200 nM in 72-h MTT assays. The cytotoxicity seen in the MTT assay proved to be apoptosis by several criteria. Interestingly, inhibition of caspase activation with the caspase inhibitor Boc-Asp(OMe)-CH(2)F reduced TUNEL labeling by 40% but did not have any effect on the loss of mitochondrial membrane potential (detected as early as 4 h after drug exposure) or cytotoxicity in MTT assays. These results suggest that the primary event in flavopiridol-induced apoptosis involves induction of mitochondrial dysfunction. Cells synchronized with aphidicolin at the G(1)-S border and treated with flavopiridol during S phase showed a marked increase in apoptosis compared with an asynchronous population or a population treated during G(2)-M. Despite the increased apoptosis, a significant proportion of synchronized cells proceeded through S, G(2)-M, and into G(1) phase in the presence of flavopiridol, demonstrating that a high-grade cell cycle arrest is not required for apoptosis. Cells synchronized at the G(1)-S border treated with a short exposure to flavopiridol also showed more than a 10-fold decrease in clonogenicity compared with asynchronous cells treated identically. CONCLUSIONS: Taken together, these data demonstrate that flavopiridol potently and selectively induces SCLC apoptosis preferentially during S phase, in a manner that involves early mitochondrial dysfunction without a requirement for a high-grade block to cell cycle progression. Furthermore, clonogenicity data suggests that prior S phase synchronization could be a highly effective way of enhancing the efficacy of bolus or short infusions of flavopiridol in the clinical setting.

Inhibition of phosphatidylinositol 3-kinase-Akt signaling blocks growth, promotes apoptosis, and enhances sensitivity of small cell lung cancer cells to chemotherapy.

A promising therapeutic alternative to inhibition of growth factor receptors is the inhibition of downstream signal transduction pathways. Such an approach may be especially important in tumors that can use signals from multiple growth factor receptors for growth and survival. Both stem cell factor (SCF) and insulin-like growth factor (IGF)-I, components of prominent small cell lung cancer (SCLC) autocrine loops, as well as FCS, can potently activate phosphatidylinositol 3-kinase (PI3K)-Akt signaling, albeit with different kinetics. SCF-induced PI3K-Akt activation occurs rapidly but fades within 60 min; IGF-I and FCS-induced activation persists for at least 6 h. SCF and IGF-I-mediated growth was potently inhibited by LY294002 in proportion to its ability to inhibit phosphatidylinositol 3-kinase (PI3K)-Akt signaling. A panel of six SCLC cell lines grown in 10% FCS was also very sensitive to LY294002, with average IC50 and LD50 of 5 and 25 microM, respectively. These drug concentrations suppressed the growth of the MRC-5 pulmonary fibroblast cell line and primary bronchial epithelial cells but did not induce significant cell death. Because LY294002 can also inhibit PI3K-related enzymes, we confirmed the role of the PI3K-Akt pathway in SCLC using doxycycline-regulated expression of a dominant-negative (kinase dead) and a constitutively active (CA; myristolated) Akt allele. Expression of dominant-negative Akt, which could only be achieved at relatively low levels, completely inhibited growth in the absence of exogenous growth factors and inhibited SCF-mediated growth but had no effect on IGF-I-mediated growth at the expression levels attained. Expression of CA Akt markedly augmented growth in the absence of exogenous growth factors but had minimal effect on growth in the presence of saturating concentrations SCF or IGF-I. Because PI3K-Akt signaling is known to promote survival under apoptotic stresses, we determined the effect of this pathway on SCLC sensitivity to etoposide. LY294002 potentiated the effect of low concentrations of etoposide in inhibiting growth and inducing apoptosis. The effect of low concentrations of LY294002 could largely be reversed by expression of CA Akt, suggesting that it was mediated by inhibition of Akt signaling. Expression of CA Akt by itself also induced resistance to etoposide-mediated apoptosis. Taken together, these data demonstrate that PI3K-Akt signaling promotes SCLC growth, survival, and chemotherapy resistance. Therefore, selective inhibitors of PI3K or Akt could potentially be useful as novel therapeutic agents in the treatment of SCLC.

Secretion of a unique peptide from interleukin-2-stimulated natural killer cells that induces endomitosis in immature human megakaryocytes.

When interleukin-2 (IL-2) was added to immature, low-ploidy (greater than 80% 2N+4N) megakaryocytes generated in IL-3 and stem cell factor (SCF)-containing liquid cultures of blood mononuclear cells highly enriched in hematopoietic progenitors, a 2- to 6-fold increase in the absolute number of polyploid (more than 8N) megakaryocytes was noted. This effect was found to be indirect and was mediated through natural killer (NK) cells that constitute the major lymphoid cell contaminating day 6 megakaryocyte cell populations. IL-2 had no effect on megakaryocytes generated from CD34(+) cells stimulated with IL-3 and SCF. However, medium conditioned by IL-2-stimulated, but not resting, NK cells (NKCM) contained a trypsin-sensitive factor capable of increasing 2- to 5-fold the number of polyploid megakaryocytes generated in vitro from IL-3 and SCF-stimulated CD34(+) cells. The activity in NKCM was dose dependent and could not be neutralized by an excess of antibodies to IL-6, IL-11, leukemia inhibitory factor (LIF), gp130, stromal cell derived factor-1a (SDF-1a), and thrombopoietin (TPO). Addition of IL-11, but not TPO, to NKCM-containing cultures resulted in further augmentation of polyploidy, with the generation of 50% to 70% polyploid megakaryocytes with a modal ploidy of 16N. This factor is distinct from TPO because it induces endomitosis in IL-3-generated megakaryocytes in vitro, whereas TPO does not, and its activity on megakaryocyte ploidy is not altered by optimal concentrations of TPO. In addition, no message for TPO is detectable in IL-2-stimulated NK cells by reverse transcription-polymerase chain reaction. These findings indicate that IL-2-stimulated NK cells produce a novel peptide, distinct from TPO, IL-6, IL-11, LIF, other gp130-associated interleukins, and SDF1a, that can induce in vitro endomitosis in immature human megakaryocytes in the presence of IL-3 and SCF.

Role of small-molecule kit receptor tyrosine kinase inhibitors in the treatment of small-cell lung cancer.

The prognosis of extensive-stage small-cell lung cancer (SCLC) has changed little in the past 3 decades, despite the documentation of high response rates to multiple chemotherapeutic regimens. Increasing the dose or the dose intensity of standard chemotherapy has increased toxicity without a significant improvement in outcome. However, our understanding of the biology of this disease has dramatically changed over the past 30 years, and this understanding has resulted in the identification of novel therapeutic targets. Evidence supporting the Kit growth factor receptor tyrosine kinase as a therapeutic target in SCLC is detailed. The properties of small-molecule Kit inhibitors currently in clinical development, as well as their efficacy in preclinical SCLC studies, are presented. The rationale for combining small-molecule Kit inhibitors with standard chemotherapy in clinical trials for extensive-stage SCLC is discussed.