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ADAM metallopeptidase domain 9 (ADAM9) is a member of the ADAM family of multifunctional, multidomain type 1 transmembrane proteins. ADAM9 is overexpressed in many cancers, including non–small cell lung, pancreatic, gastric, breast, ovarian, and colorectal cancer, but exhibits limited expression in normal tissues. A target-unbiased discovery platform based on intact tumor and progenitor cell immunizations, followed by an IHC screen, led to the identification of anti-ADAM9 antibodies with selective tumor-versus-normal tissue binding. Subsequent analysis revealed anti-ADAM9 antibodies were efficiently internalized and processed by tumor cells making ADAM9 an attractive target for antibody–drug conjugate (ADC) development. Here, we describe the preclinical evaluation of IMGC936, a novel ADC targeted against ADAM9. IMGC936 is comprised of a high-affinity humanized antibody site-specifically conjugated to DM21-C, a next-generation linker-payload that combines a maytansinoid microtubule-disrupting payload with a stable tripeptide linker, at a drug antibody ratio of approximately 2.0. In addition, the YTE mutation (M252Y/S254T/T256E) was introduced into the CH2 domain of the antibody Fc to maximize in vivo plasma half-life and exposure. IMGC936 exhibited cytotoxicity toward ADAM9-positive human tumor cell lines, as well as bystander killing, potent antitumor activity in human cell line-derived xenograft and patient-derived xenograft tumor models, and an acceptable safety profile in cynomolgus monkeys with favorable pharmacokinetic properties. Our preclinical data provide a strong scientific rationale for the further development of IMGC936 as a therapeutic candidate for the treatment of ADAM9-positive cancers. A first-in-human study of IMGC936 in patients with advanced solid tumors has been initiated (NCT04622774).

Intervention of cancer cell mitosis by antitubulin drugs is among the most effective cancer chemotherapies. However, antitubulin drugs have dose-limiting side effects due to important functions of microtubules in resting normal cells and are often rendered ineffective by rapid emergence of resistance. Antimitotic agents with different mechanisms of action and improved safety profiles are needed as new treatment options. Mitosis-specific kinesin Eg5 represents an attractive anticancer target for discovering such new antimitotic agents, because Eg5 is essential only in mitotic progression and has no roles in resting, nondividing cells. Here, we show that a novel selective Eg5 inhibitor, LY2523355, has broad target-mediated anticancer activity in vitro and in vivo. LY2523355 arrests cancer cells at mitosis and causes rapid cell death that requires sustained spindle-assembly checkpoint (SAC) activation with a required threshold concentration. In vivo efficacy of LY2523355 is highly dose/schedule-dependent, achieving complete remission in a number of xenograft tumor models, including patient-derived xenograft (PDX) tumor models. We further establish that histone-H3 phosphorylation of tumor and proliferating skin cells is a promising pharmacodynamic biomarker for in vivo anticancer activity of LY2523355. Mol Cancer Ther; 14(11); 2463–72. ©2015 AACR.

Supplementary Table 1. In vitro anticancer activity of LY2523355 against a panel of commonly used cancer cell lines; supplementary Table 2. Anticancer activity of LY2523355 in NCI-60 assay

Supplementary Data from Preclinical Evaluation of IMGC936, a Next-Generation Maytansinoid-based Antibody–drug Conjugate Targeting ADAM9-expressing Tumors

Supplementary Figure 1 (A-C). LY2523355 has potent and selective activity against microtubule-stimulated Eg5 ATPase activity but unlike antitubulins, has no effect on microtubule dynamics in vitro; supplementary Figure 2 (A-E). LY2523355 causes cell cycle arrest at mitosis with monopolar spindle and induces apoptosis; supplementary Figure 3 (A-D). Induction of apoptosis by LY2523355 requires sustained spindle assembly checkpoint (SAC)-mediated mitotic arrest; supplementary Figure 4 (A-D). Antitumor activity of LY2523355 in patient-derived xenograft (PDX) models is correlated with mitotic arrest of cancer cells; supplementary Figure 5 (A,B). Mitotic arrest and apotosis of p388 cells in ascities fluid after continuous infusion of LY2523355, demonstrating the threshold concentration effect.

Figure Legends to supplementary Figures 1-5

Table S1. Summary of most common drug-related AEs, by dosing schedule; Table S2. Summary of pharmacokinetic parameters of TAK-117 on day 1 of cycle 1 across different dosing schedules (pharmacokinetics-evaluable population); Table S3. Summary statistics of TAK-117: multiple-dose pharmacokinetic parameters across different schedules (pharmacokinetics-evaluable population).

Purpose: To evaluate the safety, MTD, pharmacokinetics, pharmacodynamics, and preliminary antitumor activity of TAK-117 (MLN1117/INK1117), an investigational PI3Kα-selective inhibitor, in patients with advanced solid tumors.Experimental Design: Seventy-one patients received oral TAK-117 once daily [100–300 mg (n = 24)] or 3 days per week [Monday–Wednesday–Friday (MWF), 200–1,200 mg (n = 27); Monday–Tuesday–Wednesday (MTuW), 200–900 mg (n = 20)], in 21-day cycles. Dose escalation proceeded via a 3 + 3 design.Results: TAK-117 once-daily dosing was associated with dose-limiting grade ≥3 alanine/aspartate aminotransferase (ALT/AST) elevations, resulting in a narrow range of tolerable doses (100–150 mg once daily). With MWF/MTuW dosing, no dose-limiting ALT/AST elevations occurred until the MTD of 900 mg; total weekly dose was 2.6-fold that of 150 mg once daily. Drug-related grade ≥3 adverse events occurred in 25%/22%/35% (including hyperglycemia in 0%/7%/15%) of once-daily/MWF/MTuW patients. TAK-117 (100–1,200 mg) exhibited moderately fast oral absorption, a generally dose proportional increase in exposure, and plasma half-life of approximately 11 hours. Total weekly exposures with 900 mg MWF/MTuW dosing were approximately 4 times greater than with 150 mg once daily. Skin pS6 expression was suppressed at ≥200 mg. There were 3/1/0 partial responses (once daily/MWF/MTuW) and 5/7/5 patients had stable disease lasting ≥3 months (all PIK3CA mutated).Conclusions: Intermittent dosing of TAK-117 had an acceptable safety profile and enabled higher doses and total weekly exposures versus once-daily dosing. Although the potential for TAK-117 as single-agent therapy appears limited, further evaluation in combination approaches for advanced solid tumors is warranted. Clin Cancer Res; 23(17); 5015–23. ©2017 AACR.

Purpose: To evaluate the safety, MTD, pharmacokinetics, pharmacodynamics, and preliminary antitumor activity of TAK-117 (MLN1117/INK1117), an investigational PI3Kα-selective inhibitor, in patients with advanced solid tumors. Experimental Design: Seventy-one patients received oral TAK-117 once daily [100–300 mg (n = 24)] or 3 days per week [Monday–Wednesday–Friday (MWF), 200–1,200 mg (n = 27); Monday–Tuesday–Wednesday (MTuW), 200–900 mg (n = 20)], in 21-day cycles. Dose escalation proceeded via a 3 + 3 design. Results: TAK-117 once-daily dosing was associated with dose-limiting grade ≥3 alanine/aspartate aminotransferase (ALT/AST) elevations, resulting in a narrow range of tolerable doses (100–150 mg once daily). With MWF/MTuW dosing, no dose-limiting ALT/AST elevations occurred until the MTD of 900 mg; total weekly dose was 2.6-fold that of 150 mg once daily. Drug-related grade ≥3 adverse events occurred in 25%/22%/35% (including hyperglycemia in 0%/7%/15%) of once-daily/MWF/MTuW patients. TAK-117 (100–1,200 mg) exhibited moderately fast oral absorption, a generally dose proportional increase in exposure, and plasma half-life of approximately 11 hours. Total weekly exposures with 900 mg MWF/MTuW dosing were approximately 4 times greater than with 150 mg once daily. Skin pS6 expression was suppressed at ≥200 mg. There were 3/1/0 partial responses (once daily/MWF/MTuW) and 5/7/5 patients had stable disease lasting ≥3 months (all PIK3CA mutated). Conclusions: Intermittent dosing of TAK-117 had an acceptable safety profile and enabled higher doses and total weekly exposures versus once-daily dosing. Although the potential for TAK-117 as single-agent therapy appears limited, further evaluation in combination approaches for advanced solid tumors is warranted. Clin Cancer Res; 23(17); 5015–23. ©2017 AACR.

Folate Receptor alpha (FRα) is an attractive antibody drug conjugate (ADC) target due to its over expression in multiple epithelial malignancies including ovarian, endometrial, triple negative breast, and non-small cell lung cancer, with limited expression on normal tissues. IMGN853 (i.e., mirvetuximab soravtansine and M9346A-sulfo-SPDB-DM4), a FRα targeting ADC, is currently in phase III (MIRASOL) clinical evaluation as monotherapy in patients with platinum-resistant epithelial ovarian cancer with high levels of FRα expression. The MIRASOL study builds on the results from the prior randomized study, FORWARD I, which demonstrated that improved outcomes with IMGN853 correlated with FRα expression, with the strongest treatment effects for all efficacy endpoints in ovarian cancer patients with FRα-high disease (Moore, ESMO 2019). In order to address the unmet needs of additional patient populations, we sought to develop a next generation FRα-targeting ADC active against tumors with a broad range of FRα expression. Development of a new molecular entity with the desired antitumor properties included optimization of the antibody format and the linker-payload. The resulting lead ADC denoted IMGN151 comprises an asymmetric, bivalent, biparatopic antibody targeting two independent epitopes of FRα, linked to the highly potent maytansinoid derivative DM21 via a stable cleavable peptide linker. The average drug per antibody ratio is 3.5. The binding, internalization and processing of the biparatopic IMGN151 and the parent monospecific antibodies were compared using 3H-antibodies. In tumor cells with medium (JHOS4) and high (KB) FRα expression the biparatopic antibody boosted antibody binding events and processing by 100% and 170%, respectively. The plasma stability of IMGN151 was tested in a cynomolgus monkey pharmacokinetic study. The stable linker increased ADC half-life by 60 hours and conjugate exposure in vivo by 40%, as compared to IMGN853. IMGN151 activity was characterized against cell lines and xenograft models with a wide range of FRα expression and compared to IMGN853. In in vitro studies, both ADCs had similar activity against FRα-high KB cells; IMGN151 was up to 200 times more active against four FRα-medium cell lines. IMGN151 had also notably stronger bystander killing activity in a mixed culture of target-positive and negative cells. In vivo IMGN151 induced complete tumor regressions of human tumor xenograft models with high (KB, H-score of 300), medium (Igrov-1 and Ishikawa, H-score of 140 and 100, respectively) and low (Ov-90, H-score of 30) FRα expression. All tested doses were well tolerated with no body weight loss observed. With a novel biparatopic antibody and linker payload design, IMGN151 has shown potent antitumor activity against ovarian cancer models with a broad range of FRα expression, which warrants further development into the clinic for patients with tumors expressing FRα at a wide range of levels. Citation Format: Olga Ab, Laura M. Bartle, Leanne Lanieri, Jose F. Ponte, Qifeng Qiu, Surina Sikka, Juliet A. Costoplus, Wayne Deats, Nicholas C. Yoder, Wayne C. Widdison, Katherine Mucciarone, Kate Selvitelli, Ying Chen, Neeraj Kohli, Thomas Chittenden, Richard Gregory, Yulius Setiady, Eric H. Westin. IMGN151 - A next generation folate receptor alpha targeting antibody drug conjugate active against tumors with low, medium and high receptor expression [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2890.

Targeting multiple hallmarks of cancer with drug combinations may provide unique opportunities for cancer therapeutics; however, phenotypic quantification is necessary to understand in vivo mechanisms of action of each drug alone or in combination. Immunohistochemistry (IHC) can quantify phenotypic changes, but traditional methods are not amenable for high-throughput drug discovery. In this article, we describe a high-content method to quantify changes in tumor angiogenesis, vascular normalization, hypoxia, tumor cell proliferation, and apoptosis using IHC. This method to quantify tumor model phenotypes can be useful for cancer drug discovery by increasing the understanding of: (i) tumor models used in efficacy studies, (ii) changes occurring during the growth of the tumor, and (iii) novel mechanisms of actions of cancer therapeutics.

This first-in-human report examined the recommended Phase 2 dose and schedule of litronesib, a selective allosteric kinesin Eg5 inhibitor. Two concurrent dose-escalation studies investigated litronesib across the dose range of 0.125–16 mg/m2/day, evaluating the following schedules of administration on a 21-day cycle: Days 1, 2, 3; Days 1, 5, 9; Days 1, 8; Days 1, 5; or Days 1, 4, with or without pegfilgrastim. Best overall response was defined per Response Evaluation Criteria in Solid Tumors (RECIST Version 1.0). Pharmacokinetic (PK) evaluations were performed. Exploratory PK/pharmacodynamic analyses investigated the relationship between litronesib plasma exposure and changes in phosphohistone H3 (pHH3) levels. One hundred and seventeen patients with advanced malignancies were enrolled. Neutropenia was the primary dose-limiting toxicity. Prophylactic pegfilgrastim reduced neutropenia frequency and severity, allowing administration of higher litronesib doses, but increases in the incidences of mucositis and stomatitis were observed. Among 86 response-evaluable patients, 2 patients (2%) achieved partial response, both on the Days 1, 2, 3 regimen (5 and 6 mg/m2/day with pegfilgrastim), and 17 patients (20%) maintained stable disease for ≥6 cycles. Dose-dependent increases in litronesib plasma exposure were observed, with minor intra- and inter-cycle accumulation, along with exposure-dependent increases in pHH3 expression in tumor and skin biopsies. On the basis of the results of these studies, two regimens were selected for Phase 2 exploration: 6 mg/m2/day on Days 1, 2, 3 plus pegfilgrastim and 8 mg/m2/day on Days 1, 5, 9 plus pegfilgrastim, both on a 21-day cycle.

Purpose This phase I study aims at assessing the safety and tolerability of LY2603618, a selective inhibitor of Checkpoint Kinase 1, in combination with pemetrexed and determining the maximum tolerable dose and the pharmacokinetic parameters. Experimental design This was an open-label, multicenter, dose-escalation study in patients with advanced solid tumors. Increasing doses of LY2603618 (40–195 mg/m2) were combined with 500 mg/m2 of pemetrexed. LY2603618 was administered on Days 1 and 9 and pemetrexed on Day 8 in a 28-day cycle. For all subsequent 21-day cycles, pemetrexed was administered on Day 1 and LY2603618 on Day 2. Antitumor activity was evaluated as per Response Evaluation Criteria in Solid Tumors 1.0. Results A total of 31 patients were enrolled into six cohorts (three at 40 mg/m2 over 4.5-hour infusion, 1-hour infusion in subsequent cohorts: three each at 40 mg/m2, 70 mg/m2, and 195 mg/m2; 13 at 105 mg/m2; six at 150 mg/m2). Four patients experienced a dose-limiting toxicity: diarrhea (105 mg/m2); reversible infusion-related reaction (150 mg/m2); thrombocytopenia (195 mg/m2); and fatigue (195 mg/m2). The maximum tolerated dose was defined as 150 mg/m2. The pharmacokinetic data demonstrated that the exposure of LY2603618 increased in a dose-dependent manner, displayed a suitable half-life for maintaining required human exposures while minimizing the intra- and inter-cycle accumulation, and was unaffected by the pemetrexed administration. The pharmacokinetic-defined biologically efficacious dose was achieved at doses ≥105 mg/m2. Conclusion LY2603618 administered approximately 24 h after pemetrexed showed acceptable safety and pharmacokinetic profiles.

Intervention of cancer cell mitosis by antitubulin drugs is among the most effective cancer chemotherapies. However, antitubulin drugs have dose-limiting side effects due to important functions of microtubules in resting normal cells and are often rendered ineffective by rapid emergence of resistance. Antimitotic agents with different mechanisms of action and improved safety profiles are needed as new treatment options. Mitosis-specific kinesin Eg5 represents an attractive anticancer target for discovering such new antimitotic agents, because Eg5 is essential only in mitotic progression and has no roles in resting, nondividing cells. Here, we show that a novel selective Eg5 inhibitor, LY2523355, has broad target-mediated anticancer activity in vitro and in vivo. LY2523355 arrests cancer cells at mitosis and causes rapid cell death that requires sustained spindle-assembly checkpoint (SAC) activation with a required threshold concentration. In vivo efficacy of LY2523355 is highly dose/schedule-dependent, achieving complete remission in a number of xenograft tumor models, including patient-derived xenograft (PDX) tumor models. We further establish that histone-H3 phosphorylation of tumor and proliferating skin cells is a promising pharmacodynamic biomarker for in vivo anticancer activity of LY2523355. Mol Cancer Ther; 14(11); 2463–72. ©2015 AACR.

A minority (less than 2%) of all lymphoproliferative disorders are derived from small T cells. These include T-cell prolymphocytic leukemia, T-cell granular lymphocytic leukemia, and mycosis fungoides/Sézary syndrome. With the possible exception of early-stage, skin-localized mycosis fungoides, all are considered incurable, although palliation can be achieved with radiation therapy, chemotherapy, biologic therapy, and combinations of these modalities. Of these disorders, mycosis fungoides is the most common; it follows an indolent, though gradually progressive, course that spans years. The T-cell prolymphocytic leukemias, in contrast, are generally refractory to treatment, with a median survival of typically less than 1 year. Although effective therapy remains elusive in most cases, the development of nucleosides as a class of chemotherapeutic agents and biologics, including interferon, monoclonal antibodies, and vitamin A derivatives, offers new hope for at least more effective palliation of these progressive lymphoproliferative disorders. However, rapid improvement in the treatment of these disorders remains hampered by the rarity of these individual entities. More rapid progress in treatment depends on national and international cooperation to accrue patients for definitive trials of sufficient size to evaluate new treatment options quickly.

Hormone-dependent breast cancer responds to primary therapies that block estrogen production or action, but tumor regrowth often occurs 12-18 months later. Additional hormonal treatments that further reduce estrogen synthesis or more effectively block its action cause additional remissions, but the mechanisms responsible for these secondary responses are not well understood. As a working hypothesis, we postulated that primary hormonal therapy induces adaptive changes, resulting in enhanced estrogen receptor (ER) expression and target gene activation and, further, that secondary treatment modalities interfere with these receptor-mediated transcriptional pathways. To test this hypothesis, we used an MCF-7 breast cancer model system involving deprivation of estradiol in culture for a prolonged period. These long-term estradiol-deprived (LTED) cells adapt by acquiring the ability to regrow in the absence of added estradiol. The experimental paradigm involved the comparison of wild-type cells with LTED cells. As endpoints, we directly assessed ER expression at the messenger RNA-, protein-, and ligand-binding levels and ER functionality by quantitating reporter gene activation and expression of endogenous estrogen target gene messenger RNA, as well as ER coactivator levels. Our data demonstrated an adaptive increase in ER expression and in basal ER functionality, as assessed by read-out of three different transfected reporters in LTED, as opposed to wild-type MCF-7 cells. Increased reporter gene read-out was dramatically inhibited by the pure antiestrogen ICI 182,780. As verification that endogenous (as well as transfected) estrogen target genes had enhanced transcription, we found that the basal levels of c-myb and c-myc message were substantially increased in LTED cells and could be inhibited by antiestrogen. Interestingly, the levels of c-myb and c-myc message in the LTED cells seemed to be increased out of proportion to the degree of ER reporter gene activation and were similar to those in wild-type cells maximally stimulated with estradiol. In addition, not all estrogen-responsive genes were activated, because transforming growth factor-alpha message level was not increased in LTED cells. Up-regulation of the steroid receptor coactivator SRC-1 did not seem to mediate the process of enhanced ER-induced transcription. Considering these observations together, we suggest that long-term estradiol deprivation causes adaptive processes that not only involve up-regulation of the ER but also influence the specificity and magnitude of activation of estrogen-responsive genes.

Despite the frequent description of 6q- structural abnormalities in human leukemias and lymphomas, rearrangements of the c-MYB locus have not been detected. We have detected a rearrangement in the c-MYB proto-oncogene in the cell line CCRF-CEM, an immature T-cell leukemia cell line which is not 6q-. Due to this rearrangement, a large portion of the c-MYB promoter conserved between the human and murine c-MYB genes is lost. The rearranged locus, which we have designated MRR (MYB rearranged region), has been cloned and mapped to chromosome 6. Field inversion gel electrophoresis (FIGE) studies reveal that the MRR sequence is linked to the c-MYB locus, suggesting that the rearrangement is due to a submicroscopic deletion. The rearrangement appears to have no effect on c-MYB promoter activity as analyzed in CCRF-CEM cells. The normal locus of the MRR sequence has been cloned from a human placental genomic library. Partial sequence analysis of this clone reveals that a portion of the DNA lost in the rearrangement shows a high degree of homology to a member of the myc family of oncogenes. Thus the characterization of this rearrangement has yielded a new set of probes for the study of chromosome 6q abnormalities in human leukemias and lymphomas and provides the first evidence for potential involvement of the c-MYB locus itself in submicroscopic deletions within chromosome 6.

We previously identified two membrane-bound folate binding proteins, FBP1 and FBP2, in murine L1210 leukemia cells. We now report on the development of two variant murine erythroleukemia cell lines that were used for direct comparison and biochemical characterization of the two murine folate binding proteins. Based on the results of northern analysis and the mobilities of affinity-labeled proteins on polyacrylamide gels, these cell lines exhibit specific up-regulated expression of FBP1 or FBP2. The affinities of the folate binding proteins for various (anti)folates were determined based upon the ability of the compounds to inhibit binding of [ 3 H]jfolic acid. The two proteins exhibited considerably different affinities and stereospecificities and, in general, FBP2 consistently bound each test compound with lesser affinity than FBP1. Both proteins displayed greatest affinity for folic acid, 5-methyltetrahydrofolate, and the antifolates CB3717 and 5,10-dideazatetrahydrofolate (DDATHF). Conversely, the proteins exhibited poor affinity for the dihydrofolate reductase inhibitors methotrexate and aminopterin. For 5-formyltetrahydrofolate, FBP1 had high affinity for the (6 S ) diastereoisomer, whereas FBP2 showed preference for the non-physiologic (6 R ) diastereoisomer. The binding properties of FBP1 and FBP2 overexpressed in these cell lines closely paralleled those of their respective human homologs. These lines provide a model system in which to examine the biochemical characteristics of the individual folate binding proteins without the potential problems associated with expression of proteins in dissimilar cell lines.

High-level expression of the c-sis oncogene, which encodes the β chain of platelet-derived growth factor, transforms immortalized rodent fibroblasts in vitro to a malignant phenotype. c-sis gene expression has been demonstrated in a variety of human tumors, although generally at levels much lower than those shown to transform cells in vitro. We examined the effect of lower levels of c-sis expression on the phenotype of NIH 3T3 fibroblasts. Clones with various levels of c-sis expression were generated by transfecting NIH 3T3 cells with a plasmid that expressed the human c-sis cDNA and the TN5 neomycin-resistance gene. G418-resistant clones, which expressed the c-sis cDNA, were selected and characterized. Alterations in the phenotype of the clones that expressed c-sis ranged from increased growth in soft agar to malignant tumor formation in nude and syngeneic mice. Increased levels of c-sis cDNA expression correlated with the acquisition of features of transformation in a dose-dependent manner and altered the cellular phenotype in a manner consistent with the progression of cells towards malignancy. These data support a model in which low levels of sis gene expression in tumors contribute to the acquisition of some features of transformation but require complementation by other genes or factors to produce a fully malignant phenotype. © 1992 Wiley-Liss, Inc.

The c-kit proto-oncogene encodes a receptor having tyrosine-specific kinase activity and has been mapped to chromosome 4 in the human and chromosome 5 in the mouse, at the dominant white spotting locus (W). Mutations at the W locus affect various aspects of murine hematopoiesis. The c-kit proto-oncogene has been shown to be expressed by leukemic myeloblasts, but not by normal unseparated human bone marrow cells. The role of this oncogene in differentiation and proliferation of human hematopoietic progenitors is presently undefined. To determine c-kit expression by normal hematopoietic progenitors, CD34+ cells were isolated from disease-free human bone marrow, and RNA-based polymerase chain reaction (PCR) techniques were used to assess expression. By this method, we have demonstrated c-kit expression by CD34+ bone marrow progenitors. To address the functional requirement for c-kit expression in normal human hematopoiesis, CD34+ cells were incubated in the presence of sense, antisense, or missense oligonucleotides to c-kit, and subsequently cultured in the presence of either recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) or recombinant human interleukin-3 (rhIL-3). Exposure of CD34+ cells to c-kit antisense oligonucleotides significantly inhibited colony-forming ability of cells cultured in the presence of rhIL-3, but had no effect on colony formation of cells cultured in rhGM- CSF. Together, these data suggest a possible role for c-kit in hematopoietic proliferation and differentiation that may be linked to some, but not all, stimulatory factors.

Bryostatin 1 is a macrocyclic lactone protein kinase C (PK-C) activator which has demonstrated promising antileukemic activity in preclinical studies. We have examined the effect of this agent on the metabolism and cytotoxicity of 1-β- d -arabinofuranosylcytosine (ara-C) in both log phase and high-density human promyelocytic leukemia cells (HL-60). Exposure of low-density cells to 12.5 nM bryostatin 1 for 24 hr prior to a 4-hr incubation with 1 or 10 μM ara-C resulted in nearly a 2-fold increase in ara-CTP formation. When cells were maintained under high-cell density conditions (e.g. 5 × 10 6 cells/ mL) for 24 hr prior to ara-C exposure, a 90% reduction in ara-CTP formation and ara-C DNA incorporation was observed. However, coincubation of high-density cells with bryostatin 1 for 24 hr increased ara-CTP formation 6- to 8-fold, yielding levels essentially equivalent to those achieved in low-density cells. Smaller (but still significant) increases in ara-C DNA incorporation were also noted. Enhancement of ara-CTP formation by bryostatin 1 occurred over a broad ara-C concentration range (0.1 to 100 μM), involved a temperature-dependent process, could not be mimicked by addition of hematopoietic growth factors, and was not related to neutralization of toxic or inhibitory substances in high-density medium. Exposure of cells to bryostatin 1 did not lead to morphologic or functional evidence of HL-60 cell maturation or an increase in cell viability, but did produce a decline in cellular proliferative activity as determined by thymidine and bromodeoxyuridine incorporation and cytofluorometric analysis. Bryostatin 1 did not exert its effects in high-density cells by inhibiting ara-C deamination or by interfering with ara-CTP dephosphorylation, but instead appeared to act by enhancing ara-C phosphorylation. Although cell-free extracts obtained from high-density cells exposed to bryostatin 1 exhibited levels of deoxycytidine kinase activity compared to controls, treated cells did display a significant decline in intracellular dCTP levels (e.g. 0.7 vs 1.3 solpmol 10 6 ), and nearly a 2-fold increase in ATP and UTP concentrations. Ara-CTP formation was also increased substantially by other PK-C activators including phorbol dibutyrate and mezerein (10–100 nM); this process was inhibited more than 70% by the PK-C inhibitor H-7 (50 μM), but not by the PK-C inhibitors staurosporine, tamoxifen, and HA1004. Finally, coadministration of ara-C and bryostatin 1 resulted in greater than expected inhibitory effects toward HL-60 cell clonogenic growth. These findings suggest that the novel agent bryostatin 1 induces biochemical perturbations in leukemic cells that favor ara-C activation, particularly in high-density cells exhibiting impaired ara-C nucleotide formation. They also raise the possibility that pharmacologie agents acting through second messenger pathways may modulate the metabolism of ara-C, and potentially other nucleoside analogs.

e22169 Background: Metrics such as RECIST and % Change from baseline (%CFB) are regularly used as efficacy end-points in Phase-1 to estimate response rate and establish dose-response relationships....

Background: As the oncology clinical research community empirically explores alternatives to RECIST criteria [ 1 , 2 ] for quantifying antitumor effects of investigational agents, it is worthwhile to investigate how accurately all of these metrics reflect the true treatment effect under the effects of clinically realistic patient heterogeneity, tumor burden measurement uncertainty, and variability in the timing of tumor burden assessments. In this work, we compare the operating characteristics of kinetic-based methods requiring additional pre-baseline assessments to the more widely used change from baseline methods (eg RECIST). Methods: We simulated tumor burden time courses in populations of patients with known magnitudes of antitumor effect, with distributions of intrinsic tumor growth rates typical of early phase oncology trials [ 1 , 3 , 4 ], and with observed CT error magnitude (8.5%) [ 5 ]. We then compared the estimated growth rate inhibition (GRI) response metric to the change from baseline (CFB) and RECIST metrics in terms of (a) dependence on intrinsic tumor growth rate (TGR) and follow up time, (b) ability to recover a dose-response relationship, (c) ability to classify “responder” and “nonresponder” patients, (d) ability to classify “good” and “bad” drugs at the trial level, and (e) implications of applying unequal costs to false positives and false negatives in (d), taking into account the incremental cost of acquiring/reading the pre-study scan for the GRI method. Results: The GRI method outperformed the CFB method in all criteria (a-e) tested on the parameter range encountered in 14 published clinical trials [ 1 , 3 , 4 ]. Conclusions: Since it is impossible to know in clinical practice the “true” treatment effect of an investigational drug, simulations are needed to compare the fidelity of novel response metrics to %CFB and RECIST criteria. Under the assumptions used in this simulation study, the GRI method outperforms CFB metrics to a magnitude sufficient to offset the cost of the additional pre-study scan.

The SH2 and SH3 binding partner AFAP-110 is a tyrosine phosphorylated substrate of Src. AFAP-110 has been hypothesized to link Src to actin filaments, which may contribute to the effects of Src upon actin filament integrity. However, it has been unclear what effect activated Src (Src527F) has upon AFAP-110 structure or function and whether AFAP-110 plays a role in actin filament integrity. We report here that the carboxy terminal 127 amino acids of AFAP-110 are comprised of an alpha-helical region that contains a leucine zipper motif. This indicated the potential of AFAP-110 to self-associate. Expression of the carboxy terminus as a fusion protein (GST-cterm) will permit affinity absorption of cellular AFAP-110. The integrity of the alpha-helical leucine zipper motif in GST-cterm is required for affinity absorption, but binding is not due to a classical leucine zipper interaction. Co-expression of Src527F, unlike cSrc, will abrogate affinity absorption of AFAP-110 with GST-cterm. These data indicate that Src527F has affected a change in the carboxy terminal structure that renders AFAP-110 unavailable for affinity absorption. Superose chromatography demonstrate that AFAP-110 will fractionate as a monomer or multimer, indicating AFAP-110 can be detected in a self-associated form in cell lysates. Co-expression of Src527F resulted in AFAP-110 fractionating with a molecular weight that predicts only a multimeric population. Deletional mutagenesis also indicate a biological role for the carboxy terminus in cellular localization and actin filament integrity. Deletion of the entire carboxy terminal alpha-helix (84 amino acids) will not permit AFAP-110 to efficiently colocalize with actin filaments or the cell membrane. Deletion of only the leucine zipper region of the carboxy terminal alpha-helix (44 amino acids) from AFAP-110 (AFAPAdeltazip) demonstrate that both AFAPdeltalzip and actin filaments are repositioned into rosette-like structures, similar to the effects of Src527F, while co-expression of AFAP-110 with cSrc will not affect actin filaments. These data indicate that AFAP-110 can play an important role in modulating actin filament integrity through carboxy terminal interactions that can be affected by Src527F.

Background: MLN0128 is an investigational, potent, and highly selective inhibitor of mTORC1/2, which are integral to cell proliferation, angiogenesis, and cellular metabolism. This first-in-human study (NCT01058707) aimed to determine the maximum tolerated dose (MTD), dose-limiting toxicity (DLT), safety, pharmacokinetics (PK), pharmacodynamics (PD), and preliminary antitumor activity of oral MLN0128. Methods: Pts aged ≥18 years with advanced solid tumors were enrolled in a 3+3 dose-escalation design initially to receive MLN0128 once daily (QD), and then enrolled in escalating intermittent schedules of once weekly (QW), 3 days/week (QDx3d QW), or 5 days/week (QDx5d QW) dosing, in 28-day cycles. Blood samples were collected at multiple timepoints for PK analysis. PD endpoints were evaluated in skin to determine the effect on mTORC1/2-dependent biomarkers. A preclinical PK-efficacy model was generated (Phoenix NLME v1.1) with tumor xenograft efficacy data, and implemented using clinical PK parameters to simulate tumor volume-time curves for various MLN0128 doses/schedules. Response was assessed by RECIST v1.1. Results: 115 pts received MLN0128 doses in the ranges 2-7 mg QD (n=30), 7-40 mg QW (n=30), 6-20 mg QDx3d QW (n=33), and 7-13 mg QDx5d QW (n=22). Median age was 60 years (range 24-89); 40% were male. The most common tumor types were colorectal (22%), renal (9%), and ovarian (8%) cancer. The MTDs were 6 mg QD, 40 mg QW, 16 mg QDx3d QW, and 10 mg QDx5d QW. Based on the overall safety profiles, the recommended phase 2 doses were 5 mg QD, 40 mg QW, 9 mg QDx3d QW, and 7 mg QDx5d QW. Pts received a median of 2 cycles (range 1-23). The most common drug-related adverse events (AEs) were hyperglycemia (65%), nausea (60%), vomiting (44%), decreased appetite (36%), diarrhea (33%), asthenia (30%), and mucosal inflammation (30%). The most common drug-related grade ≥3 AEs were hyperglycemia (12%), asthenia (9%), and mucosal inflammation (5%). MLN0128 exhibited dose-linear PK with a plasma half-life of ∼8 h, and did not accumulate in plasma with QD dosing. Modeling of preclinical PK-efficacy data and simulation of human tumor volume-time curves using clinical PK parameters suggested potential greater antitumor effect with schedules using more frequent dosing (i.e. QD, QDx5d QW). There was a treatment-related inhibition of mTORC1/2 biomarkers in skin. Two of 10 pts (20%) with renal cancer receiving MLN0128 at 15 and 40 mg QW had a partial response; 5 pts (6%) had stable disease for ≥6 cycles. Conclusions: Based on the safety profile, PK/PD, simulated tumor volume-time curves, and responses, MLN0128 5 mg QD and 40 mg QW were selected for further evaluation in an expansion phase in pts with renal, endometrial, or urothelial cancer. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C252. Citation Format: Jeffrey R. Infante, Josep Tabernero, Andres Cervantes, Shadia Jalal, Howard A. Burris, Teresa Macarulla, J. Alejandro Perez-Fidalgo, Rachel Neuwirth, Chirag Patel, Esha Gangolli, Rachael Brake, Jeffrey Sturm, Eric H. Westin, Michael Gordon. A phase 1, dose-escalation study of MLN0128, an investigational oral mammalian target of rapamycin complex 1/2 (mTORC1/2) catalytic inhibitor, in patients (pts) with advanced non-hematologic malignancies. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C252.

Schwann cells are the primary cell type in the disfiguring lesions associated with neurofibromatosis type 1 (NF-1). These lesions also contain abnormally high numbers of mast cells, a cell type which develops in response to stem cell factor. We report here that neonatal and adult rat and human Schwann cells, as well as a transfected rat Schwann cell line and a human Schwannoma line derived from an NF-1 patient, all produced stem cell factor mRNA and protein. In coculture experiments, surface expression of stem cell factor by neonatal rat Schwann cells was profoundly downregulated by contact with dorsal root ganglion neurites. The receptor for stem cell factor, KIT, was not expressed in normal Schwann cells but was expressed in the human Schwannoma line, suggesting that aberrant KIT expression may form an autocrine loop in certain Schwann cell neoplasias. © 1994 Wiley-Liss, Inc.

Circulating tumor cells have been shown to be prognostic of survival in metastatic breast, colon, and prostate cancers. Additionally, CTCs are of interest because they may be representative of the phenotype/genotype of the primary and metastatic tumors, and may be a useful tool (i.e. patient stratification) for drug development. CTCs, as “liquid biopsies” are potentially useful as a PD marker as it allows easy repeat sampling before and after drug treatment. We describe the development of a CTC assay that measures the induction of pHH3 by LY2523355, a selective small molecule inhibitor of the human Eg5 kinesin. pHH3 is a biomarker of mitosis. In tumor xenograft mouse models, LY2523355 treatment was shown to reduce tumor volume with elevation of pHH3 in tumors. The CTC assay was developed using the FDA cleared Veridex/CellSearch™ instrument and reagents for enumeration of CTCs from blood. CellSearch defines CTCs as EpCam+/DAPI+/CK 8, 18, 19+/CD45−. For assay development, cells from tumor cell lines representing major solid tumor types were chosen, cultured, treated with LY2523355, or with various standard-of-care chemotherapeutics, and spiked into whole blood drawn into CellSave™ (preservative+EDTA). For initial assay development, mouse whole blood was used and results reproduced subsequently with human whole blood from healthy subjects. The spiked tumor cells in blood samples were recovered using the CellSearch Mouse/Rat CTC kit (for mouse blood) or human CXC kit (for human blood), supplemented with the R-PE conjugated anti-pHH3 antibody. pHH3 in the recovered tumor cells was detected in the open/fourth channel on the CellSearch instrument. About 1–4% of all cultured tumor cells were positive for pHH3 without drug treatment. Significant induction of pHH3 (30–50% cells) in sensitive tumor cell lines was observed with 24 hour treatment with only drugs interfering with mitosis (paclitaxel, taxotere, and LY2523355). The magnitude of the induction of pHH3 in the cells after treatment was dose and cell-line dependent. A dose response of pHH3 induction was observed with 0.5nM-25nM of LY2523355 with several cultured tumor cell lines (Colo205, HCT116, H441, SKBR3, MDA-MB-468), spiked into blood, and assayed using CellSearch. The concentrations of the dose response are similar to the drug exposure levels of that of preclinical tumor xenograft studies. LY2523355 is currently in early phase clinical development, and monitoring CTCs with pHH3 expression post-treatment may be a useful PD marker. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A47.

Background: LY2603618 is a selective inhibitor of Chk1, a protein kinase that plays a key role in the DNA damage checkpoint. Inhibition of Chk1 is predicted to enhance the effects of antimetabolites, such as gemcitabine. Methods: This study is a Phase 1–2 study in patients with solid tumors (Phase 1) and advanced pancreatic adenocarcinoma (Phase 2). In Phase 1, gemcitabine (1000 mg/m2) was administered on Days 1, 8, and 15 of a 28-day cycle. LY2603618 was administered on Days 2, 9, and 16. Patients were assessed for safety, tolerability, and dose-limiting toxicity (DLT). A recommended Phase 2 dose (RP2D) was determined based on safety, dose intensity, and pharmacokinetics (PK). Results: A total of 50 patients were enrolled. Patients were treated at 70 (n =3), 105 (n=3), 150 (n=7), 200 (n=11), 250 (n=6) mg/m2and at 2 additional flat-fixed dose cohorts of 200 (n=10) and 230 (n=10) mg. The most frequent AEs reported included fatigue, thrombocytopenia, anemia, nausea, neutropenia, and constipation, which are consistent with those reported with gemcitabine monotherapy. During escalation, DLTs included neutropenia, infusion-related reaction and thrombocytopenia, with thrombocytopenia being dose limiting. The maximum tolerated dose (MTD) was determined to be 200 mg/m2. The systemic exposure of LY2603618 increased in a dose-dependent manner and the LY2603618 systemic clearance was dose-independent across all doses on average. The mean LY2603618 half-life varied across doses but was consistent with a half-life (i.e., >10 hr and 21,000 ng hr/mL and Cmax > 2000 ng/mL) were achieved by all but one patient. Based on safety/tolerability, the ability to maintain dose intensity, and PK, a RP2D of 230 mg was selected. A total of 17 of 30 patients received more than 2 cycles of therapy. Conclusions: LY2603618 administered in combination with gemcitabine demonstrated an acceptable safety profile; the MTD for this regimen was defined at 200 mg/m2 in the originally designed study. However at a fixed dose of 230 mg, LY2603618 in combination with gemcitabine had an acceptable safety profile and the observed exposures exceed those required for biological effect in nonclinical models. This dose is being evaluated in the Phase 2 component of the study. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A94.

Circulating tumor cells are prognostic of survival in metastatic breast, colon, and prostate cancers. Additionally, CTCs are of interest because they may be representative of the phenotype/genotype of the primary and metastatic tumors, and a useful tool (e.g. patient stratification) for drug development. CTCs, as “liquid biopsies” are potentially useful as a pharmacodynamic marker allowing easy repeat sampling before and after drug treatment. We describe the development of a CTC assay measuring H2AX induction, a marker of DNA damage. The CTC assay was developed using the FDA cleared Veridex/CellSearch™ instrument and reagents for enumeration of CTCs from blood. CellSearch defines CTCs as EpCam+/DAPI+/CK 8,18,19+/CD45−. For assay development, cells from tumor cell lines representing major solid tumor types were chosen, cultured, treated with inhibitors of checkpoint kinase 1 (Chk1, LY2603618, LY2606368) or with various standard-of-care (SOC) chemotherapeutics. These treated cells were then spiked into whole blood drawn into CellSave™ (preservative+EDTA). LY2603618 and LY2606368 are inhibitors of Chk1 kinase currently in clinical development. LY2603618 is meant to be used in combination with a DNA damaging agent, while LY2606368 has potent single agent activity. Inhibition of Chk1 in cells with DNA damage allows the cells to progress into mitosis without DNA repair, leading to cell death. In tumor xenograft mouse models, treatment reduced tumor volume with elevation of γH2AX in tumors and anagen hair follicles at drug exposure levels similar to concentrations used for the CTC γH2AX development assay. For initial assay development, mouse whole blood was used and results reproduced subsequently with human whole blood from healthy subjects. The spiked tumor cells in blood samples were recovered using the CellSearch Mouse/Rat CTC kit (for mouse blood) or human CXC kit (for human blood), supplemented with the R-PE conjugated anti-γH2AX antibody. γH2AX in the recovered tumor cells was detected in the open/fourth channel on the CellSearch instrument. Results were confirmed by flow analysis. Without drug treatment, only about 0.2–2% of all cultured tumor cells were γH2AX positive. The magnitude of the induction of γH2AX in the cells after treatment was dose and cell-line dependent. Significant induction of H2AX (>20% cells) was observed after 24 hour treatment with only a few SOC agents (cisplatin, etoposide) in sensitive tumor cell lines; the majority induced moderate to low levels (usually Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A49.

Background: LY2523355 is a potent allosteric inhibitor of the motor protein Eg5 which selectively inhibits bipolar mitotic spindle formation. The objective of this Phase 1 study was to determine a Phase 2 dose and schedule of LY2523355 that may be safely administered to patients with advanced malignancy. Methods: Study I1Y-MC-JFBA is an open-label, multicenter, dose-escalation study in patients with advanced malignancy. A modified 3+3 dose-escalation scheme was used, followed by a dose confirmation phase with pre- and post-treatment tumor biopsies. Patients received LY2523355 as a 1hr infusion on days 1, 2 and 3 of a 21-day cycle with determination of the MTD with and without pegfilgrastim administration on day 4. Patients were assessed every other cycle by RECIST. Treatment related PD changes in phosphohistone H3 (pHH3) and Eg5 expression were assessed in skin and tumor biopsies by immunohistochemistry. Results: 46 patients were enrolled in the study as of October 2010. The most frequent related AEs reported (>10%) include leukopenia, neutropenia, fatigue, nausea, stomatitis, vomiting, alopecia, anemia, diarrhea, lymphopenia, pruritis/itching and rash. Dose-limiting toxicities include increased INR, leukopenia, diarrhea, rash (palmar plantar erythrodysesthesia), febrile neutropenia, and mucositis. The single and multiple dose pharmacokinetics of LY2523355 and metabolite were characterized. Dose dependent increase in LY2523355 plasma exposure and a minor degree of intra- and inter-cycle accumulation were observed. An exposure-dependent increase in pHH3 and Eg5 expression and increase in apoptotic cells in tumor and skin biopsies has been demonstrated. One patient with ovarian cancer had a confirmed partial response. Nine other patients had stable disease for at least 2 cycles. Conclusions: The MTD of LY2523355 for this schedule has been established with and without pegfilgrastim at 6mg/m2/d and 4 mg/m2/d, respectively. These doses have shown a quantifiable PD response in skin and tumor biopsies confirming the importance of maximizing exposure intensity for maximum PD response. Dose confirmation is on-going. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A86.

2600 Background: LY2523355 is an allosteric inhibitor of Eg5 which selectively inhibits bipolar mitotic spindle formation. The objective of this Phase 1 study is to determine the Phase 2 dose and s...

A. Nasir, R.D. Van Horn, B. Domazet, X.S. Ye, K.M. Credille, A. Kyshtoobayeva, J.T. Brandt, E.H. Westin, A.E. McGlothlin, T.R. Holzer. Eli Lilly and Co., Diagnostic and Experimental Medicine, Indianapolis IN, USA; Eli Lilly and Co., Lilly Research Labs, Indianapolis IN, USA; Clarient, Immunopathology, Aliso Viejo CA, USA; Eli Lilly and Co., Oncology Business Unit, Indianapolis IN, USA; Eli Lilly and Co., Statistics, Indianapolis IN, USA

Antitubulin agents including taxanes and vincas that target mitosis of rapidly dividing cancer cells are among the most effective cancer therapies in current clinical use. However, these antitubulin agents also have debilitating side effects that are dose-limiting, such as neuropathy, due to their disruption of the normal microtubule functions in resting cells including neuronal cells. Eg5 is an evolutionarily conserved mitosis-specific kinesin essential for bipolar mitotic spindle formation and has no roles in microtubule functions of resting cells. Inactivation of Eg5 causes mitotic arrest of proliferating cells, resulting in formation of monopolar spindles. Targeting Eg5 for cancer treatment thus represents an attractive strategy that has the potential to maximize the anticancer efficacy by inhibiting cancer cell mitosis while minimizing debilitating side effects associated with antitubulins. Here we describe a selective ATP-non competitive small molecule inhibitor of human Eg5 kinesin. The Eg5 inhibitor shows no effects on microtubule dynamics in cell-free assays and arrests cells specifically at mitosis with monopolar spindles, resulting in rapid cancer cell death. Growth inhibition assays against a panel of 21 cancer cell lines shows that the Eg5 inhibitor has potent and broad-spectrum activity with IC50 values ranged from 0.55 nM to 14.2 nM. Quantitative live cell imaging and high content imaging reveal that the Eg5 inhibitor has a threshold concentration activity and kills cancer cells specifically at mitosis in a time/cell cycle, but not concentration above the threshold,-dependent manner. Consistent with the in vitro activities, the Eg5 inhibitor shows broad-spectrum antitumor activity in preclinical xenograft tumor models representing major human cancer histologies also including drug resistant tumors and demonstrates superiority as compared to several chemotherapeutic agents targeting G2/M. Furthermore, its in vivo antitumor activity is highly schedule-dependent with a clear threshold dose effect, as expected from in vitro observations. Indeed, the Eg5 inhibitor exhibits a robust PK/PD relationship in antitumor activity and its antitumor activity is associated with mitotic arrest of cancer cells and subsequent cell death. The Eg5 inhibitor is currently being evaluated in Phase I studies. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A62.

Interference of DNA damage checkpoints has been demonstrated to be a highly effective means of increasing the cytotoxicity of a wide number of anti-cancer therapies. Ionizing radiation, DNA cross-linkers, topoisomerase inhibitors and anti-metabolites all cause severe cellular DNA damage and activation of multiple DNA damage checkpoints. Cell cycle arrest at these checkpoints protects injured cells from apoptotic cell death until the DNA damage can be repaired. In the absence of functioning DNA damage checkpoints, cells with damaged DNA proceed into premature mitosis followed by rapid apoptotic death. A key protein kinase involved in activating and maintaining the S and G2/M checkpoints is Chk1. Pharmacological inhibition of Chk1 in the absence of p53 leads to abrogation of the DNA damage checkpoints and has been shown to enhance the preclinical activity of many standard of care chemotherapeutic agents. LCI-1 is a novel small molecule inhibitor of Chk1 (IC50 = 7 nM). In cell-based experiments LCI-1 inhibited doxorubicin-dependent autophosphorylation of Chk1 (IC50 = 52 nM). Treatment of cells with LCI-1 alone caused a phenotype identical to that reported for cells depleted of Chk1, demonstrating the mechanism-dependent activity of the compound. Treated with 100 nM doxorubicin, HeLa cells arrested at the G2M checkpoint. When treated 24 hours later with LCI- 1, the arrested cells traversed the G2M checkpoint, allowing cells to proceed into mitosis with unresolved replicated chromosomes. Consistent with abrogation of the Chk1-dependent G2M checkpoint, HT-29 cells (mutant p53) treated with LCI-1 were more sensitive to killing by gemcitabine then were LCI-1 treated HCT116 cells (wild-type p53). In vivo, LCI-1 effectively inhibited Chk1 autophosphorylation, as well as released the S and G2M block induced by gemcitabine treatment. When used preclinically in combination with DNA damaging agents, LCI-1 increased DNA damage and cell death over gemcitabine alone. These results indicate that LCI-1 may prove to be an effective potentiator of DNA damaging therapies in the clinic. LCI-1 is currently undergoing clinical testing in combination with gemcitabine and pemetrexed. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B248.

This study was conducted to examine the cell surface changes associated with oncogene induced transformation. Using the transfection technique the DNA of a human acute lymphocytic leukemia (ALL) was used to transform murine NIH3T3 cells. Balb/c mice were immunized with these transfectants and their immune splenocytes were used to produce a monoclonal antibody (17-9H3). Antibody 17-9H3 was demonstrated to bind to the cell membranes of transfectants and leukemia cells but not normal 3T3 cells in an enzyme linked immunosorbent assay. Fresh human leukemias, cultured leukemia lines and normal hemopoietic cells were examined with immunoperoxidase staining techniques to determine the specificity of 17-9H3. Our data suggest that the antigen associated with a human acute lymphocytic leukemia oncogene is ubiquitous, distributed among both neoplastic and normal hemopoietic cells. Among fresh human leukemias the antigen appears to be present primarily on fresh null ALLs and chronic myelogenous leukemia (CML) in blast crisis. This antigen was also found to be expressed by the majority of cultured T-cell lines tested.

Human T-cell leukemia-lymphoma virus (HTLV) is a type C retrovirus associated with a subtype of mature T-cell malignancy in humans. HTLV also infects normal human cord blood mature T lymphocytes in vitro and induces a number of phenotypic changes in these cells, including their continuous growth and partial or complete independence of T-cell growth factor (TCGF). As part of our initial study designed to analyze gene(s) specifically activated by HTLV infection, we have isolated a recombinant DNA clone by differential screening of a cDNA library made from mRNA of a human T-cell lymphoma cell line producing HTLV. This cDNA identifies a single-copy gene in all human DNAs and a single mRNA species of 2.3 kilobases expressed at several hundred copies per cell in five HTLV-positive neoplastic T-cell lines. In addition, cord blood T lymphocytes infected with HTLV, but not the uninfected counterparts, express high levels of mRNA from this gene. A survey of different human hematopoietic cell types showed that this gene is expressed at low or undetectable levels (less than 10 copies) in human T, B, myeloid, or erythroid cell lines; in moderate amounts in lymphoid precursor (immature) cell lines; and in high amounts in lectin-activated mature T-cells, comparable to those of HTLV-infected T-cell lines. The precise function of this gene has not yet been determined.

The nucleotide sequence of a transforming human c-sis complementary DNA shows an open reading frame 723 base pairs in length located downstream from an in-phase terminator thymine-guanine-adenine codon. Sequences within this region were identical to those previously determined for the exons of the normal human c-sis gene. Thus, the predicted transforming product, a protein of 27,281 daltons, may be the actual precursor for normal human platelet-derived growth factor chain A.

The mechanism of leukaemogenk transformation by human T-cell leukaemia/Iymphoma virus (HTLV), a retrovirus implicated in the aetiology of certain adult T-cell leukaemias and lymphomas, is unknown but is conceivably associated with the expression of the cellular analogues of retroviral oncogenes. The HUT-102 cell line1, derived from a cutaneous T-cell lymphoma and infected with HTLV2–4, expresses several cellular oncogenes5. It is unusual among haemopoietic cell lines in that one of these is c-sis, the gene from which the oncogene v-sis of the simian sarcoma virus was derived, and perhaps the gene for platelet-derived growth factor (PDGF)6,7. To explore the possible role of c-sis expression in HTLV-induced disease, we have obtained cDNA clones of c-sis from HUT-102 cells. Here we describe two such clones and report that one of them transforms NIH-3T3 cells. This is the first example of transformation of NIH-3T3 cells by a human onc gene other than c-ras or Blym, as well as the first demonstration of transformation by a human cDNA clone.

We have karyotyped cells from a lymph node of a patient with atypical lymphoid hyperplasia. Among other clonal chromosomal abnormalities, a t(2;19) translocation was observed with breakpoints at 2p11.2 and 19q13. The genes for transforming growth factor alpha and beta have been mapped to 2p11-p13 and 19q13, respectively, but Southern blot analysis did not reveal any alteration in the structure of these genes. Similarly, the kappa immunoglobulin gene, which maps to 2p11-p12 was not rearranged. In addition, Southern blot analysis using immunoglobulin and T-cell receptor genes as probes, did not demonstrate any clonality of either B or T cells. We propose that this patient represents an early, polyclonal stage of atypical hyperplasia. The chromosome changes observed may have been one of the etiologic factors causing this disorder.

The transforming genes of retroviruses (v-onc) are derived from normal cellular genes referred to as proto-oncogenes. These cellular genes have the capacity for conversion to oncogenes (c-onc) that are capable of inducing or maintaining the transformed state when they are overly expressed or altered by mutation or rearrangement. To study the possible involvement of these genes in human leukemia, we have analyzed their expression in a variety of fresh samples. We found that a number of oncogenes are expressed in different leukemic types and that although the transcript size did not vary for each gene, the copy number did. The myc gene (2.4 kb transcript) and the rasHa gene (1.5 kb transcript) were universally expressed. But in contrast to rasHa, the myc signal intensity varied. Myb (4.5 kb transcript) was expressed in all samples except B cell diseases. We detected low levels of abl expression (multiple mRNA species) in all leukemic types analyzed. Sis gene (4.2 kb transcript) expression was restricted to one patient sample with chronic myelogenous leukemia in blast transformation.

Solid tumors, particularly those involving the colon, breast, and lung, are the most common tumors in humans. However, many technical difficulties exist in obtaining analyzable chromosomes from these tumors, including the inability to stimulate cell division. Phorbol-12,13-dibutyrate (PDBu) is a tumor promoter that activates a variety of cellular responses, including proliferation. Using flow cytometry, we have demonstrated that PDBu acts as a mitogen in primary cultures of colon tumor cells. Based on these results, we developed a short-term culture technique that greatly improves the yield of analyzable metaphases from colon tumors. Stimulated cultures consistently contained at least ten times more metaphases than unstimulated cultures, and chromosome morphology was improved. By modifying this technique with the addition of the calcium ionophore A23187, we have successfully obtained analyzable chromosomes from the peripheral blood of normal individuals, chronic lymphocytic leukemia patients, and a nodular small cell lymphoma patient. These results demonstrate that mitogenic stimulation by PDBu is a valuable technique in the cytogenetic analysis of colon tumors. By using PDBu alone or in combination with other agents, this technique may also be applicable to many other tumors that are difficult to karyotype because of an inability to obtain mitoses.

The amino acid sequence of human mast cell tryptase was determined from corresponding cDNA cloned from a lambda ZAP library made with mRNA derived from a human mast cell preparation. Tryptase is the major neutral protease present in human mast cells and serves as a specific marker of mast cells by immunohistologic techniques and as a specific indicator of mast cell activation when detected in biologic fluids. Based on nucleic acid sequence, human tryptase consists of a 244-amino acid catalytic portion of 27,423 D with two putative N-linked carbohydrate binding sites and a 30-amino acid leader sequence of 3,048 D. A His74, Asp120, Ser223 catalytic triad and four cystine groups were identified by analogy to other serine proteases. Regions of amino acid sequence that are highly conserved in serine proteases, in general, were conserved in tryptase. The catalytic portion of human tryptase had an 84% amino acid sequence similarity with that of dog tryptase; their leader sequences had a 67% similarity. Asp217 in the substrate binding pocket of human tryptase is consistent with a specificity for Arg and Lys residues at the site of cleavage (P1), whereas Glu245 is consistent with the known preference of human tryptase for substrates with Arg or Lys also at P3, analogous residues also being present in dog tryptase. Asp244, which is substituted for the Gly found in dog tryptase and in most serine proteases, is present in the putative substrate binding pocket and may confer additional substrate specificity on human tryptase for basic residues. Further studies now can be designed to elucidate these structure-function relationships.

Bovine leukemia virus (BLV), an exogenous retrovirus of cattle [5, 11], induces B-lymphocyte neoplasms (termed enzootic bovine leukosis, EBL) after long latent periods [2]. BLV contains no host cellular sequences and does not appear so far to bear genes capable of inducing transformation directly. A wide range of genomic sites can accomodate BLV proviruses. Transcription of viral DNA including long terminal repeated sequences has not been detected, strongly suggesting that viral gene expression is not required for maintenance of the tumor state. No expression of 3’-proximate cellular sequences has been observed, indicating that proximate downstream promotion did not take place in the cases examined [12].

Polyadenylated RNAs of certain human tumour cell lines are shown to contain transcripts related to the cell-derived transforming onc genes of molecularly cloned primate, murine or avian transforming retrovirus genomes. Thus, analogues of retroviral transforming genes are both present and frequently expressed in human neoplastic cells.

Both enhanced and altered expressions of cellular oncogenes have recently been implicated in specific forms of human malignancy (for review: [2, 8]). The fact that certain human tumors have characteristic chromosomal translocations has led to the hypothesis that specific cellular oncogenes may be 'activated' in these genetic recombinations. In a number of human undifferentiated B-cell lymphoma (UBL) cell lines carrying the t(8:14) translocation it has been shown that the human c-myc gene is located on the region of chromosome 8 (8q24), which is translocated to the immunoglobulin heavy-chain (IHC) locus on chromosome 14 [7, 24]. We report here the molecular cloning of the recombination site between c-myc and IHC in fresh uncultured cells obtained from a patient with rapidly progressive and fatal acute lymphoblastic leukemia. The translocation was associated with an enhanced c-myc expression in the tumor cells of this patient.

Chromosome translocations involving 8q24, the band to which c-myc has been mapped (Dalla-Favera et al., 1982), are a uniform finding in Burkitt's lymphoma (Bernheim et al., 1981). However, in only a minority of the tumors is the rearrangement of the c-myc locus sufficiently close to the gene to be detected with currently available probes (Dalla-Favera et al., 1983). Approximately 25% of diffuse large-cell lymphomas have also been reported to have translocations involving 8q24 (Mitelman, 1985), but there have been no reports of c-myc rearrangements in this form of non-Hodgkin's lymphoma. We have examined the structure of the c-myc locus in primary tumor tissue of 10 cases of diffuse large-cell lymphoma. In one patient, Southern blot analysis revealed additional c-myc fragments in the tumor DNA but not in the germ-line DNA. Southern blot analysis using probes from both the heavy- and light-chain immunoglobin loci showed that the myc rearrangement was unlikely to involve the immunoglobulin loci in this patient.

Total cellular RNAs from a variety of fresh and culture-derived human hematopoietic neoplastic cell types at various stages of differentiation and human sarcoma, carcinoma, melanoma, and glioblastoma cell lines were enriched for poly(A)- containing sequences, fractionated by gel electrophoresis, and blot hybridized to a cloned DNA probe containing the transforming sequences (v-amv) of avian myeloblastosis virus (AMV), a virus known to cause myeloid leukemias in chickens. Expression of RNA sequences homologous to AMV was detected in all immature myeloid and lymphoid T cells in addition to the single erythroid cell line examined, but not in mature T cells or in B cells, including lymphoblast cell lines derived from patients with Burkitt lymphoma. In addition, induction of the cell line HL60, a promyelocytic leukemia line, to differentiate with dimethyl sulfoxide or retinoic acid resulted in a reduction of the level of expression of the human cellular gene c-amv homologous to v-amv. There was no detectable c-amv mRNA in any of the solid tumor cell lines examined. Thus, expression of the human c-amv gene could be correlated with the stage of differentiation of different hematopoietic cell types determined by morphologic and marker studies. Expression of c-amv could not be correlated with the extent of methylation in HL60 and in HL60 induced to differentiate with dimethyl sulfoxide.

Substantial evidence indicates that retroviral transforming (v-onc) genes originated by means of recombination between a present nontransforming virus and normal cellular sequences (Duesberg et al., this volume). These sequences, called cellular onc gene, are highly conserved during evolution, suggesting that they may code for protein products which are essential for cell growth or tissue differentiation. As these normal cellular genes are homologous to viral- transforming genes, their potential role in tumorigenesis is of great interest. As an alternative to direct transformation by a viral onc gene, abnormal activation of a cellular onc gene may cause transformation. Two models have been proposed for such a mechanism. First, high levels of expression of a cellular onc gene may be caused by the insertion nearby of a viral promoter [12, 15, 16, 17] or by alteration of the physiological promoter by a mutagenic agent such as a chemical carcinogen. Secondly, a cellular onc gene may be relocated in a transcriptionally active region of the genome as a consequence of chromosomal rearrangements [2, 5, 6, 13]. In this chapter we review the evidence for a possible third mechanism for onc gene activation in neoplastic cells, that of gene amplification. The human homologue, c-myc, of the transforming gene of avian myelocytomatosis virus (MC29), which is expressed at relatively high levels in the human promyelocytic leukemia cell line HL-60, is stably amplified in the genome of these cells [7]. Amplification was also detected in primary, uncultured leukemic cells from the same individual, suggesting that the c-myc amplification may have been involved in the leukemic transformation in this case.