Solid Tumor Xenografts Research Articles

Initial Testing (Stage 1) of MK-8242-A Novel MDM2 Inhibitor-by the Pediatric Preclinical Testing Program.

MK-8242 is an inhibitor of MDM2 that stabilizes the tumor suppressor TP53 and induces growth arrest or apoptosis downstream of TP53 induction. MK-8242 was tested against the Pediatric Preclinical Testing Program (PPTP) in vitro cell line panel at concentrations from 1.0 nM to 10.0 μM and against the PPTP in vivo xenograft panels using oral gavage on Days 1-5 and Day 15-19 at a dose of 125 mg/kg (solid tumors) or 75 mg/kg (acute lymphoblastic leukemia [ALL] models). The median IC50 for MK-8242 was 0.07 μM for TP53 wild-type cell lines versus >10 μM for TP53 mutant cell lines. MK-8242 induced a twofold or greater delay in time to event in 10 of 17 (59%) of TP53 wild-type solid tumor xenografts, excluding osteosarcoma xenografts that have very low TP53 expression. Objective responses were observed in seven solid tumor xenografts representing multiple histotypes. For the systemic-disease ALL panel, among eight xenografts there were two complete responses (CRs) and six partial responses (PRs). Two additional MLL-rearranged xenografts (MV4;11 and RS4;11) grown subcutaneously showed maintained CR and PR, respectively. The expected pharmacodynamic responses to TP53 activation were observed in TP53 wild-type models treated with MK-8242. Pharmacokinetic analysis showed that MK-8242 drug exposure in SCID mice appears to exceed that was observed in adult phase 1 trials. MK-8242-induced tumor regressions across multiple solid tumor histotypes and induced CRs or PRs for most ALL xenografts. This activity was observed at MK-8242 drug exposures that appear to exceed those observed in human phase 1 trials.

511. Engineered Donor Marrow Macrophages Phagocytose Cancer Cells and Aggressively Shrink Solid Tumor Xenografts Compared to Tumor Associated Macrophages

Cell-based immunotherapies such as those based on engineered T-cells appear safe and often effective against liquid tumors. In solid tumors, macrophages are typically abundant, but the density of tumor associated macrophages (TAMs) correlates with poor clinical outcomes as they promote tumor growth, immunosuppression, and are nonphagocytic. In our studies, less differentiated donor marrow phagocytes are engineered to target tumors and selectively phagocytose cancer cells. Xenograft tumors were made on the flanks of NSG mice using a tdTomato human lung carcinoma cell line (A549). Systemic injections of anti-human IgG (anti-hum) with large tumors (~70 mm2) showed no effect on tumor growth. However, systemic injection of bone marrow from donor NSG mice together with biweekly anti-hum treatments effectively stopped growth of the solid tumors. Replacing anti-hum with a non-specific antibody had no effect on tumor growth. Based on tdTomato signal intensity within macrophages isolated from tumors, 10-fold more donor macrophages are phagocytic compared to resident TAMs (2-3% are phagocytic). Since cancer cells express on their surface ‘self’ markers that limit the phagocytosis of these cells, we inhibited the ‘self’ receptors on the injected donor phagocytes prior to systemic injection of the donor marrow. This combination of ‘self’-receptor inhibition with anti-hum causes a rapid decrease in tumor burden, shrinking tumors by ~40% in just 10 days compared to a similar growth of untreated tumors in the same time period. The anti-hum injection was again necessary as injection of a non-specific antibody failed to affect tumor growth. Tumor analysis showed that >85% of macrophages that were ‘self’-receptor inhibited had phagocytosed the tdTomato A549 cells, which is ~30-fold greater than resident macrophages. Importantly, these cell therapy treatments appear safe with no significant decreases in hematocrit or platelets, which is unlike the anemia that has been reported upon systemic injection of ‘self’ inhibitors. Our results thus suggest that therapies based on engineered macrophages can be safe and effective against solid tumors if three requirements are met: a phagocytic phenotype, target opsonization, and inhibition of ‘self’ signaling.

A fully human CXCR4 antibody demonstrates diagnostic utility and therapeutic efficacy in solid tumor xenografts.

For physiologically important cancer therapeutic targets, use of non-invasive imaging for therapeutic guidance and monitoring may improve outcomes for treated patients. The CXC chemokine receptor 4 (CXCR4) is overexpressed in many cancers including non-small cell lung cancer (NSCLC) and triple negative breast cancer (TNBC). CXCR4 overexpression contributes to tumor growth, progression and metastasis. There are several CXCR4-targeted therapeutic agents currently in clinical trials. Since CXCR4 is also crucial for normal biological functions, its prolonged inhibition could lead to unwanted toxicities. While CXCR4-targeted imaging agents and inhibitors have been reported and evaluated independently, there are currently no studies demonstrating CXCR4-targeted imaging for therapeutic guidance.Monoclonal antibodies (mAbs) are commonly used for cancer therapy and imaging. Here, an 89Zr-labeled human CXCR4-mAb (89Zr-CXCR4-mAb) was evaluated for detection of CXCR4 expression with positron emission tomography (PET) while its native unmodified analogue was evaluated for therapy in relevant models of NSCLC and TNBC. In vitro and in vivo evaluation of 89Zr-CXCR4-mAb showed enhanced uptake in NSCLC xenografts with a high expression of CXCR4. It also had the ability to detect lymph node metastases in an experimental model of metastatic TNBC. Treatment of high and low CXCR4 expressing NSCLC and TNBC xenografts with CXCR4-mAb demonstrated a therapeutic response correlating with the expression of CXCR4. Considering the key role of CXCR4 in normal biological functions, our results suggest that combination of 89Zr-CXCR4-mAb-PET with non-radiolabeled mAb therapy may provide a precision medicine approach for selecting patients with tumors that are likely to be responsive to this treatment.

Azacyclic FTY720 Analogues That Limit Nutrient Transporter Expression but Lack S1P Receptor Activity and Negative Chronotropic Effects Offer a Novel and Effective Strategy to Kill Cancer Cells in Vivo.

FTY720 sequesters lymphocytes in secondary lymphoid organs through effects on sphingosine-1-phosphate (S1P) receptors. However, at higher doses than are required for immunosuppression, FTY720 also functions as an anticancer agent in multiple animal models. Our published work indicates that the anticancer effects of FTY720 do not depend on actions at S1P receptors but instead stem from FTY720s ability to restrict access to extracellular nutrients by down-regulating nutrient transporter proteins. This result was significant because S1P receptor activation is responsible for FTY720s dose-limiting toxicity, bradycardia, that prevents its use in cancer patients. Here, we describe diastereomeric and enantiomeric 3- and 4-C-aryl 2-hydroxymethyl pyrrolidines that are more active than the previously known analogues. Of importance is that these compounds fail to activate S1P1 or S1P3 receptors in vivo but retain inhibitory effects on nutrient transporter proteins and anticancer activity in solid tumor xenograft models. Our studies reaffirm that the anticancer activity of FTY720 does not depend upon S1P receptor activation and uphold the promise of using S1P receptor-inactive azacyclic FTY720 analogues in human cancer patients.

Engineered Donor Marrow Macrophages Phagocytose Cancer Cells and Aggressively Shrink Solid Tumor Xenografts Compared to Tumor Associated Macrophages

Cell-based immunotherapies such as those based on engineered T-cells appear safe and often effective against liquid tumors. In solid tumors, macrophages are typically abundant, but the density of tumor associated macrophages (TAMs) correlates with poor clinical outcomes as they promote tumor growth and immunosuppression. In our studies, less differentiated donor marrow phagocytes are engineered in order to target tumors and selectively phagocytose cancer cells. Xenograft tumors were first made on the flanks of NSG mice using a tdTomato human lung carcinoma cell line (A549). Systemic injections of anti-human IgG (anti-hum) with large tumors (~70 mm2) showed no effect on tumor growth. However, systemic injection of bone marrow from donor NSG mice together with biweekly anti-hum treatments effectively stopped growth of the solid tumors. Replacing anti-hum with a non-specific antibody had no effect on tumor growth. Based on tdTomato signal intensity within macrophages isolated from tumors, 5-10-fold more donor macrophages are phagocytic compared to resident TAMs (2-3% are phagocytic). Since cancer cells express on their surface 'self' markers that limit the phagocytosis of these cells, we inhibited the 'self' receptors on the injected donor phagocytes prior to systemic injection of the donor marrow. This combination of 'self'-receptor inhibition with anti-hum causes a rapid decrease in tumor burden, shrinking tumors by ~40% in just 10 days compared to a similar growth of untreated tumors in the same time period. The anti-hum injection was again necessary as injection of a non-specific antibody failed to affect tumor growth. Tumor analysis showed that >65% of macrophages that were 'self'-receptor inhibited had phagocytosed the tdTomato A549 cells, which is ~20-fold greater than resident macrophages. Importantly, these cell therapy treatments appear safe with no significant decreases in hematocrit or platelets, which is unlike the anemia that has been reported upon systemic injection of 'self' inhibitors. Our results thus suggest that therapies based on engineered macrophages can be safe and effective against solid tumors if three requirements are met: a phagocytic phenotype, target opsonization, and inhibition of 'self' signaling. Future experiments will involve characterizing macrophage phenotypes of donor versus resident macrophages as well as efforts to maintain the necessary phagocytic phenotype. DisclosuresNo relevant conflicts of interest to declare.

Abstract B170: Discovery of tubulysin payloads for antibody drug conjugates with potent in vitro activity and in vivo efficacy in solid tumor models

Abstract Antibody drug conjugates (ADCs) combine the specificity of antibodies with the potency of small molecule cytotoxic drugs and have the potential to provide significant efficacy as a treatment for cancer. The objective of this work was to identify potent new cytotoxic ADC payloads that can be used to target diverse tumor types. Here we report for the first time the discovery of fully synthetic tubulysin payloads which belong to a class of highly cytotoxic natural products that disrupt the cellular microtubule network leading to apoptosis of tumor cells. Our fully synthetic tubulysin payloads are comprised of: (i) a tubulysin warhead that displays pM potency, (ii) a protease cleavable amino-acid sequence and (iii) a tether bearing a reactive maleimide group. Tubulysin-based ADCs were generated via site-specific conjugation of these payloads to cysteines engineered into antibodies against cancer antigen target oncofetal protein 5T4. The resulting ADCs showed potent in vitro cell killing and in vivo efficacy in multiple solid tumor xenograft models including prostate cancer, non-small cell lung adenocarcinoma, breast cancer and gastric carcinoma. Furthermore, specific structural features of the tubulysin warhead, linker design and antibody engineering were shown to impact the overall in vitro and in vivo properties of the ADCs. Thus, these synthetic tubulysin payloads represent novel microtubule network disrupting compounds that display potent preclinical anti-tumor activity as an ADC that could be advanced to the clinic. Citation Format: Dorin Toader, Jay Harper, Chris Lloyd, Rose Marwood, David Bannister, Shenlan Mao, Cui (Tracy) Chen, Haihon (Helen) Zhong, Vahe Bedian, Fengjiang Wang, Lakshmaiah Gingipalli, Melisa Vasbinder, Pamela Thompson, Ryan Fleming, Byniam Bezabeh, Nazzareno Dimasi, Changshou Gao, Adeela Kamal. Discovery of tubulysin payloads for antibody drug conjugates with potent in vitro activity and in vivo efficacy in solid tumor models. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B170.

Initial Testing of NSC 750854, a Novel Purine Analog, Against Pediatric Tumor Models by the Pediatric Preclinical Testing Program.

NSC 750854 is a purine analog with an antitumor activity profile distinctive from that of other anticancer purines. It has shown significant activity against adult cancer preclinical models. NSC 750854 was tested against the Pediatric Preclinical Testing Program (PPTP) in vitro cell line panel at concentrations from 1.0 nM to 10 μM and against the PPTP in vivo xenograft panels administered intraperitoneally at a dose of 5 mg/kg daily for 5 days repeated at day 15. The median relative IC50 (rIC50 ) value for the PPTP cell lines was 32 nM (range from 11 to 124 nM), with consistent cytotoxicity across all cell lines. Acute lymphoblastic leukemia (ALL) cell lines were more sensitive to NSC 750854 than non-ALL cell lines. NSC 750854 induced significant differences in EFS distribution compared to control in 31 of 35 (89%) solid tumor xenografts. It induced tumor growth inhibition meeting criteria for intermediate or high event free survival (EFS) T/C activity in 17 of 32 (53%) evaluable solid tumor xenografts (most consistently in the rhabdomyosarcoma panel). Objective responses were observed in 15 of 37 (41%) solid tumor xenografts and in all eight leukemia models with complete response (CR) or maintained complete response (MCR) in seven of eight leukemia models. NSC 750854 has a unique spectrum of antitumor activity compared with other agents tested by the PPTP as it induces regression in tumor models with limited sensitivity to most agents tested to date. Given the promising level of activity observed for NSC 750854 against PPTP preclinical models, further exploration of its mechanism of action is warranted.

Thiarabine, 1-(4-Thio-β-D-arabinofuranosyl)cytosine. A Deoxycytidine Analog With Excellent Anticancer Activity.

Thiarabine has demonstrated exceptional antitumor activity against numerous human tumor xenografts in mice, being superior to gemcitabine, clofarabine, or cytarabine. Unlike cytarabine, thiarabine demonstrated excellent activity against solid tumor xenografts, suggesting that this agent has the kind of robust activity in animal models that leads to clinical utility. Thiarabine is effective orally (bioavailability of approximately 16%) and with once per day dosing: Two characteristics that distinguish it from cytarabine. Although both the structure and basic mechanism of action of thiarabine are similar to that of cytarabine, there are many quantitative differences in the biochemical pharmacology of these two agents that can explain the superior antitumor activity of thiarabine. Two important attributes are the long retention time of the 5'-triphosphate of thiarabine in tumor cells and its potent inhibition of DNA synthesis. The biochemical pharmacology of thiarabine is also different from that of gemcitabine. Thiarabine has been evaluated in three phase I clinical trials, where it has demonstrated some activity in heavily pretreated patients with hematologic malignancies and solid tumors. Because of its impressive activity against numerous human tumor xenografts in mice, its unique biochemical activity, and encouraging clinical results in phase I clinical trials, we believe thiarabine should continue to be evaluated in the clinic for treatment of hematologic and/or solid tumors. The preclinical results to date (superior in vivo antitumor activity, oral bioavailability, and once per day dosing), suggest that thiarabine could replace cytarabine in the treatment of acute myelogenous leukemia.

Pharmacodynamic and genomic markers associated with response to the XPO1/CRM1 inhibitor selinexor (KPT-330): A report from the pediatric preclinical testing program.

Selinexor (KPT-330) is an inhibitor of the major nuclear export receptor, exportin 1 (XPO1, also termed chromosome region maintenance 1, CRM1) that has demonstrated activity in preclinical models and clinical activity against several solid and hematological cancers. Selinexor was tested against the Pediatric Preclinical Testing Program (PPTP) in vitro cell line panel at concentrations from 1.0 nM to 10 μM and against the PPTP in vivo xenograft panels administered orally at a dose of 10 mg/kg thrice weekly for 4 weeks. Selinexor demonstrated cytotoxic activity in vitro, with a median relative IC50 value of 123 nM (range 13.0 nM to >10 μM). Selinexor induced significant differences in event-free survival (EFS) distribution in 29 of 38 (76%) of the evaluable solid tumor xenografts and in five of eight (63%) of the evaluable ALL xenografts. Objective responses (partial or complete responses, PR/CR) were observed for 4 of 38 solid tumor xenografts including Wilms tumor, medulloblastoma (n = 2), and ependymoma models. For the ALL panel, two of eight (25%) xenografts achieved either CR or maintained CR. Two responding xenografts had FBXW7 mutations at R465 and two had SMARCA4 mutations. Selinexor induced p53, p21, and cleaved PARP in several solid tumor models. Selinexor induced regression against several solid tumor and ALL xenografts and slowed tumor growth in a larger number of models. Pharmacodynamic effects for XPO1 inhibition were noted. Defining the relationship between selinexor systemic exposures in mice and humans will be important in assessing the clinical relevance of these results.

Abstract 1615: Initial testing (stage 1) of the Curaxin, CBL0137, by the Pediatric Preclinical Testing Program (PPTP)

Abstract The Curaxin, CBL0137, is a member of a new class of small molecules that simultaneously activate p53 and inhibit cancer-associated stress response pathways such as NFκB and Heat Shock Factor 1. CBL0137 sequesters the FACT (FAcilitates Chromatin Transcription) complex on chromatin, inhibiting its function. CBL0137 has shown antitumor activity against a broad range of tumor models. Because of its novel mechanism of action CBL0137 was evaluated for its antitumor activity against the PPTP in vitro and in vivo models. Methods: CBL0137 was tested against the PPTP's in vitro cell line panel (n = 23) at concentrations ranging from 1.0 nM to 10.0 μM using the PPTP's standard 96 hour exposure period. CBL0137 was tested against the PPTP solid tumor xenografts using a dose of 50 mg/kg administered by the intravenous route weekly for 4 weeks. Standard PPTP time to event and objective response metrics were used to assess CBL0137 in vivo activity. Results: The median relative IC50 (rIC50) value for the PPTP cell lines was 0.28 μM, with a range from 0.13 μM to 0.80 μM. There were no significant differences in rIC50 values by histotype. The median rIC50 for the neuroblastoma cell lines exceeded that of the non-neuroblastoma cell lines (0.51 μM vs 0.26 μM, respectively, p = 0.16), while the median rIC50 for the ALL cell lines was less than that of the non-ALL cell lines (0.20 μM vs 0.31 μM, respectively, p = 0.06). All PPTP cell lines showed a pronounced cytotoxic effect, with T/C Ymin% values approaching 0% and with Relative I/O% values approaching -100% for all cell lines at the higher concentrations tested. CBL0137 was generally well tolerated in vivo with a 3.1% toxicity rate in the treated groups compared to a 0.8% rate for control animals. CBL0137 induced significant differences in EFS distribution compared to control in 9 of 30 (30%) of the solid tumor xenografts evaluable for this measure and in 8 of 8 (100%) evaluable ALL xenografts. Significance differences in EFS distributon were observed in 4 of 6 osteosarcoma lines, 2 of 2 rhabdoid tumor lines, and 2 of 6 rhabdomyosarcoma lines. No objective responses were observed among the 30 solid tumor xenografts. For the ALL panel, 1 xenograft achieved CR and 4 achieved PR. In summary, CBL0137 showed potent cytotoxic activity in vitro without clear histotype selectivity. In vivo the most consistent activity for CBL0137 was observed against the ALL xenografts, with most solid tumor xenograft lines showing limited response to single agent CBL0137. In considering clinical translation of these testing results, it will be important to relate the drug levels in SCID (soid tumor models) or NOD/SCID (ALL models) mice at 50 mg/kg to those tolerated in humans at the CBL0137 recommended phase 2 dose. Citation Format: Malcolm A. Smith, Min Kang, Patrick C. Reynolds, Richard B. Lock, Hernan Carol, Richard Gorlick, Anders E. Kolb, John M. Maris, Stephen T. Keir, Catherine A. Billups, Raushan Kurmasheva, Peter J. Houghton. Initial testing (stage 1) of the Curaxin, CBL0137, by the Pediatric Preclinical Testing Program (PPTP). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1615. doi:10.1158/1538-7445.AM2015-1615

Abstract 1616: Pharmacodynamic and genomic markers associated with response to the XPO1/CRM1 inhibitor selinexor (KPT-330): a report from the Pediatric Preclinical Testing Program

Abstract Background: Selinexor (KPT-330) is an inhibitor of the major nuclear export receptor, exportin 1 (XPO1, also termed chromosome region maintenance 1 or CRM1) that has demonstrated activity in preclinical models of several solid and hematological cancers. Procedures: Selinexor was tested against the PPTP in vivo xenograft panels administered orally at a dose of 10 mg/kg (3 times per week) for 4 weeks. For collection of specimens for pharmacodynamic testing, KT-10 tumors that regressed rapidly after treatment with selinexor were harvested 24 hours after a single dose of drug (10 mg/kg). Other, less responsive tumors, were harvested 2 hours after dose 6 (MWF dosing) at 10 mg/kg/dose. Immunoblots were probed for p53, p21, PARP and cleaved PARP and XPO1/CRM1. IHC analysis was performed for a comparable set of proteins to assess nuclear localization. For exome sequencing, all mutations were verified and assessed as somatic using a virtual normal subtraction algorithm. Results: Objective responses (CRs) to selinexor were observed for 3 of 38 solid tumor xenografts including Wilms tumor (KT-10), medulloblastoma (BT-50) and ependymoma (BT-41) xenografts. For the ALL panel, 2 of 8 (25%) xenografts achieved either CR or MCR (ALL-8 and ALL-19). The mutation profiles for models with objective responses for which exome sequencing data were available (KT-10, BT-50, ALL-8 and ALL-19) were examined. The only recurring mutation was for FBXW7, with known oncogenic mutations identified for both BT-50 (R465H) and ALL-8 (R465C). One additional tested xenograft (ALL-31) had an FBXW7 mutation, but it was in the N-terminal region in the nuclear localization signal domain (c.45_46insCCT; p.15_16TR>TLR). KT-10 has a PALB2 mutation that leads to defective homologous recombination and to sensitivity to PARP inhibition. Selinexor induced a robust increase in p53 in KT-10 xenografts with PUMA induction and cleavage of PARP that was apparent after the first dose of drug, consistent with the rapid response of this tumor to treatment. Models that showed growth delay rather than regression showed a lesser induction of p53 and showed increased nuclear localization of a number of proteins (e.g., FOXO1, NFκB, and p53). Conclusions: Selinexor induced regression against several solid tumor and ALL xenografts, and slowed tumor growth for a larger number of models. Pharmacodynamic effects expected for XPO1 inhibition were noted in models without regard to tumor regression, although p53 induction was most robust in a highly sensitive Wilms tumor model. Exome sequencing identified potential genomic alterations (FBXW7 mutation and mutation in a homologous recombination gene) associated with sensitivity that require validation in a larger, independent set of tumors. Citation Format: Malcolm A. Smith, Richard Lock, Hernan Carol, John M. Maris, Richard Gorlick, E. Anders Kolb, Stephen T. Keir, Jianrong Wu, Yosef Landesman, Sharon Shacham, Dmitry Lyalin, Raushan T. Kurmasheva, Peter J. Houghton. Pharmacodynamic and genomic markers associated with response to the XPO1/CRM1 inhibitor selinexor (KPT-330): a report from the Pediatric Preclinical Testing Program. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1616. doi:10.1158/1538-7445.AM2015-1616

CC-223, a Potent and Selective Inhibitor of mTOR Kinase: In Vitro and In Vivo Characterization.

mTOR is a serine/threonine kinase that regulates cell growth, metabolism, proliferation, and survival. mTOR complex-1 (mTORC1) and mTOR complex-2 (mTORC2) are critical mediators of the PI3K-AKT pathway, which is frequently mutated in many cancers, leading to hyperactivation of mTOR signaling. Although rapamycin analogues, allosteric inhibitors that target only the mTORC1 complex, have shown some clinical activity, it is hypothesized that mTOR kinase inhibitors, blocking both mTORC1 and mTORC2 signaling, will have expanded therapeutic potential. Here, we describe the preclinical characterization of CC-223. CC-223 is a potent, selective, and orally bioavailable inhibitor of mTOR kinase, demonstrating inhibition of mTORC1 (pS6RP and p4EBP1) and mTORC2 [pAKT(S473)] in cellular systems. Growth inhibitory activity was demonstrated in hematologic and solid tumor cell lines. mTOR kinase inhibition in cells, by CC-223, resulted in more complete inhibition of the mTOR pathway biomarkers and improved antiproliferative activity as compared with rapamycin. Growth inhibitory activity and apoptosis was demonstrated in a panel of hematologic cancer cell lines. Correlative analysis revealed that IRF4 expression level associates with resistance, whereas mTOR pathway activation seems to associate with sensitivity. Treatment with CC-223 afforded in vivo tumor biomarker inhibition in tumor-bearing mice, after a single oral dose. CC-223 exhibited dose-dependent tumor growth inhibition in multiple solid tumor xenografts. Significant inhibition of mTOR pathway markers pS6RP and pAKT in CC-223-treated tumors suggests that the observed antitumor activity of CC-223 was mediated through inhibition of both mTORC1 and mTORC2. CC-223 is currently in phase I clinical trials.

21. Non-CD3-Based Chimeric Antigen Receptor (CARs) with Enhanced Anti-Tumor Activity in Solid Tumors

Chimeric antigen receptors (CARs) based upon a single chimeric molecule bearing an antigen binding domain linked in cis to the cytoplasmic domains of CD3ζ and costimulatory receptors CD28 or 4-1BB provide a potent method for engineering T cell cytotoxicity towards tumors. We describe a simple chimeric receptor based upon a killer immunoglobulin-like receptors (KIRs) normally expressed by natural killer (NK) cells and T cells. Constructed by fusing a single chain variable fragment (scFv) to the transmembrane and cytoplasmic domain of a KIR, we show that a KIR-based CAR targeting multiple tumor-related antigens triggers antigen-specific cytotoxic activity and cytokine production that is comparable to CD3ζ-based CARs with antigen-induced proliferation that is independent of additional costimulation. Using a solid tumor xenograft model that is highly resistant to CD3ζ-based CAR-targeted T cells, we further demonstrate that KIR-based CAR-targeted T cells can induce tumor regression despite in vivo persistence of CD3ζ-based CAR-modified T cells with CD28 and 4-1BB costimulatory domains. We show that the enhanced activity of the KIR-based CAR is associated with less tumor-induced T cell hypofunction that has previously been demonstrated with CD3ζ-based CARs. These data support the future clinical evaluation of a KIR-based CARs especially within solid tumors.

Abstract P2-04-05: Modeling luminal breast cancer heterogeneity: Combination therapies to suppress hormone receptor-negative subpopulations among receptor-positive ones

Abstract All Luminal breast cancers are heterogeneous, containing substantial numbers of estrogen (ER) and progesterone (PR) receptor-negative cells among the ER+PR+ ones. We have identified two such Luminal-derived ER–PR– cells: The first, we call "Luminobasal" (LB), are ER–PR– and cytokeratin 5 (CK5)-positive. The second, we call "Double Negative" (DN), are ER–PR– and CK5–. Currently, neither is targeted for treatment. Luminobasal cells: To address the relationships between true Luminal ER+PR+CK5– and Luminobasal ER–PR–CK5+ cells in Luminal cancers and tightly control their ratios we generated isogenic pure Luminal (pLUM) and pure Luminobasal (pLB) cells from solid tumor xenografts starting from the same parental T47Dco Luminal human breast cancer cell line. Cells were gene profiled and unique immunohistochemical (IHC) biomarkers for each were confirmed. Interaction dynamics studies show that in mixed-cell 3D colonies, pLUM or Luminal MCF-7 cells suppress growth of pLB cells. Similarly, in mixed-cell solid tumor xenografts, pLUM cells suppress pLB proliferation. Alarmingly, in mixed-cell models, monotherapy of a pLUM or MCF-7 subpopulation with the antiestrogen Fulvestrant inadvertently expands the number of pLB cells. This suggested that pLB cells also need to be treated. An 89 drug FDA-approved oncology library and high throughput screening methods were therefore used to show that pLB cells are specifically targeted by the EGFR inhibitors Gefitinib and Erlotinib. We then showed, in both mixed-cell 3D colonies and mixed-cell solid mouse tumors, that combination therapy using Fulvestrant plus Gefitinib constitutes a robust treatment strategy that targets both cell populations simultaneously. Double Negative cells: DN cells are EGFR– and therefore unaffected by EGFRi. However they express the Luminal marker Claudin-3 (CLD3). Among other conditions, DN cells arise as a discrete CLD3+ subpopulation within CLD3– pLB xenografts. This allowed us to use CLD3 for FACS isolation and purification of DN cells from xenografts. Purified cells were gene expression profiled, which showed that DN cells have a unique genomic signature that distinguishes them from LUM and LB cells. They express diagnostic markers suitable for IHC and FACS-sorting, some of which also present potential therapeutic targets. We propose that heterogeneous Luminal breast cancers may be best treated with combination therapies that include endocrine, EGFRi and/or DN inhibitors. Importantly, optimizing combination regimens that eradicate or suppress not only ER+PR+ cells but also diverse ER–PR– cells in Luminal disease requires that primary tumors be pre-screened for appropriate biomarkers, including but not limited to ER, PR, CK5 and EGFR. Citation Format: Allison L Scaling, Aaron J Knox, Maurcio P Pinto, Brian S Bliesner, James M Haughian, Hany A Abdel-Hafiz, Kathryn B Horwitz. Modeling luminal breast cancer heterogeneity: Combination therapies to suppress hormone receptor-negative subpopulations among receptor-positive ones [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P2-04-05.

Anticancer activity of streptochlorin, a novel antineoplastic agent, in cholangiocarcinoma

BackgroundThe aim of this study is to investigate the anticancer activity of streptochlorin, a novel antineoplastic agent, in cholangiocarcinoma.MethodsThe anticancer activity of streptochlorin was evaluated in vitro in various cholangiocarcinoma cell lines for apoptosis, proliferation, invasiveness, and expression of various protein levels. A liver metastasis model was prepared by splenic injection of HuCC-T1 cholangiocarcinoma cells using a BALB/c nude mouse model to study the systemic antimetastatic efficacy of streptochlorin 5 mg/kg at 8 weeks. The antitumor efficacy of subcutaneously injected streptochlorin was also assessed using a solid tumor xenograft model of SNU478 cells for 22 days in the BALB/c nude mouse.ResultsStreptochlorin inhibited growth and secretion of vascular endothelial growth factor by cholangiocarcinoma cells in a dose-dependent manner and induced apoptosis in vitro. In addition, streptochlorin effectively inhibited invasion and migration of cholangiocarcinoma cells. Secretion of vascular endothelial growth factor and activity of matrix metalloproteinase-9 in cholangiocarcinoma cells were also suppressed by treatment with streptochlorin. Streptochlorin effectively regulated metastasis of HuCC-T1 cells in a mouse model of liver metastasis. In a tumor xenograft study using SNU478 cells, streptochlorin significantly inhibited tumor growth without changes in body weight when compared with the control.ConclusionThese results reveal that streptochlorin is a promising chemotherapeutic agent to the treatment of cholangiocarcinoma.

Initial testing (stage 1) of the tubulin binding agent nanoparticle albumin-bound (nab) paclitaxel (Abraxane(®)) by the Pediatric Preclinical Testing Program (PPTP).

Nanoparticle albumin-bound paclitaxel (nab-paclitaxel, Abraxane(®)) is FDA approved for the treatment of several adult cancers. Antimitotic agents are essential components for curative therapy of pediatric solid tumors, although taxanes have shown limited activity. Because of the novel formulation, nab-paclitaxel was evaluated against a limited series of Pediatric Preclinical Testing Program (PPTP) solid tumors. Nab-paclitaxel was tested against a limited subset of PPTP solid tumor xenograft models at a dose of 50 mg/kg using a q4d × 3 schedule intravenously. Nab-paclitaxel was well tolerated in vivo, producing maximum weight loss of approximately 10% with recovery to baseline weight in the week following the third dose. All 20 xenograft models tested were considered evaluable for efficacy. Nab-paclitaxel induced statistically significant differences in event-free survival (EFS) distribution compared to control in 19 of 20 (95%) of the solid tumors. Objective responses were observed in 12 of 20 (60%) solid tumor xenografts. Complete responses (CR) or maintained CR were observed in 5 of 8 Ewing sarcoma models and 6 of 8 rhabdomyosarcomas. There were no objective regressions in either neuroblastoma (n = 2) or osteosarcoma (n = 2) xenograft panels. At the dose tested, systemic exposures of nab-paclitaxel in mice were somewhat greater than those tolerated in humans. The high level of activity observed against the rhabdomyosarcoma and Ewing sarcoma PPTP preclinical models makes nab-paclitaxel an interesting agent to consider for pediatric evaluation.

BMS-871: a novel orally active pan-Notch inhibitor as an anticancer agent.

This Letter describes synthesis, SAR, and biological activity of (2-oxo-1,4-benzodiazepin-3-yl)-succinamides as inhibitors of γ-secretase mediated signaling of Notch receptors. Optimization of this series led to the identification of BMS-871 (compound 30) which displayed robust in vivo efficacy in Notch-dependent leukemia and solid tumor xenograft models.

Discovery of Clinical Candidate BMS-906024: A Potent Pan-Notch Inhibitor for the Treatment of Leukemia and Solid Tumors.

Structure-activity relationships in a series of (2-oxo-1,4-benzodiazepin-3-yl)-succinamides identified highly potent inhibitors of γ-secretase mediated signaling of Notch1/2/3/4 receptors. On the basis of its robust in vivo efficacy at tolerated doses in Notch driven leukemia and solid tumor xenograft models, 12 (BMS-906024) was selected as a candidate for clinical evaluation.

Activity of MM-398, nanoliposomal irinotecan (nal-IRI), in Ewing's family tumor xenografts is associated with high exposure of tumor to drug and high SLFN11 expression.

To determine the pharmacokinetics and the antitumor activity in pediatric cancer models of MM-398, a nanoliposomal irinotecan (nal-IRI). Mouse plasma and tissue pharmacokinetics of nal-IRI and the current clinical formulation of irinotecan were characterized. In vivo activity of irinotecan and nal-IRI was compared in xenograft models (3 each in nu/nu mice) of Ewing's sarcoma family of tumors (EFT), neuroblastoma (NB), and rhabdomyosarcoma (RMS). SLFN11 expression was assessed by Affymetrix HuEx arrays, Taqman RT-PCR, and immunoblotting. Plasma and tumor concentrations of irinotecan and SN-38 (active metabolite) were approximately 10-fold higher for nal-IRI than for irinotecan. Two doses of NAL-IRI (10 mg/kg/dose) achieved complete responses maintained for >100 days in 24 of 27 EFT-xenografted mice. Event-free survival for mice with RMS and NB was significantly shorter than for EFT. High SLFN11 expression has been reported to correlate with sensitivity to DNA damaging agents; median SLFN11 mRNA expression was >100-fold greater in both EFT cell lines and primary tumors compared with NB or RMS cell lines or primary tumors. Cytotoxicity of SN-38 inversely correlated with SLFN11 mRNA expression in 20 EFT cell lines. In pediatric solid tumor xenografts, nal-IRI demonstrated higher systemic and tumor exposures to SN-38 and improved antitumor activity compared with the current clinical formulation of irinotecan. Clinical studies of nal-IRI in pediatric solid tumors (especially EFT) and correlative studies to determine if SLFN11 expression can serve as a biomarker to predict nal-IRI clinical activity are warranted.

Discovery of potent, orally bioavailable, small-molecule inhibitors of WNT signaling from a cell-based pathway screen.

WNT signaling is frequently deregulated in malignancy, particularly in colon cancer, and plays a key role in the generation and maintenance of cancer stem cells. We report the discovery and optimization of a 3,4,5-trisubstituted pyridine 9 using a high-throughput cell-based reporter assay of WNT pathway activity. We demonstrate a twisted conformation about the pyridine–piperidine bond of 9 by small-molecule X-ray crystallography. Medicinal chemistry optimization to maintain this twisted conformation, cognisant of physicochemical properties likely to maintain good cell permeability, led to 74 (CCT251545), a potent small-molecule inhibitor of WNT signaling with good oral pharmacokinetics. We demonstrate inhibition of WNT pathway activity in a solid human tumor xenograft model with evidence for tumor growth inhibition following oral dosing. This work provides a successful example of hypothesis-driven medicinal chemistry optimization from a singleton hit against a cell-based pathway assay without knowledge of the biochemical target.