Significant Differences In EFS Distribution Research Articles

Abstract LB-136: Pediatric Preclinical Testing Consortium evaluation of a DLL3-targeted antibody drug conjugate rovalpituzumab tesirine, in neuroblastoma

Abstract Introduction: Rova-T (SC16LD6.5) is an ADC targeting delta-like protein 3 (DLL3), a member of the delta-notch signaling pathway, composed of the monoclonal antibody SC16 conjugated to the DNA-damaging D6.5 pyrrolobenzodiazepine (PBD) dimer toxin. In clinical trials, Rova-T demonstrated anti-tumor activity in patients with small cell lung cancer with high DLL3 protein expression. We have identified DLL3 as highly expressed in NBs, especially the MYCN-amplified subset. Here, we characterize the expression of DLL3 in NB patient derived xenografts (PDX), and define the efficacy of Rova-T in these models ex vivo and in vivo. Methods: To assess DLL3 protein expression, we performed immunohistochemistry (IHC) on 32 PDXs from high-risk human primary tumors in tissue microarrays. We quantified cell surface DLL3 from dissociated PDX tumors using immunoblotting and flow-cytometry with a PE-conjugated DLL3 antibody. The cytotoxic activity of Rova-T against 8 dissociated PDXs placed in short term culture was evaluated using cell viability assays and a testing range of 10 fm to 10 nM. The in vivo activity of Rova-T was tested against 3 NB PDX models (Felix-PDX, COG-N-452x and COG-N-415x) using single intraperitoneal injection on day 0. Treatment groups included: vehicle control, IgGADC6.5 at 0.6 mg/kg, and Rova-T at 0.1 mg/kg, 0.3 mg/kg or 0.6 mg/kg. Events were defined as quadrupling of tumor volume from day 0. Results: DLL3 protein was expressed in 27 of 32 NB PDXs. The median percentage of tumor cells with a ≥ 2+ intensity was 25%-55% in 12/27 samples, 55%-85% in 8/27 samples and 85%-100% in 7/27 samples. The relative IHC scores in the tested samples correlated with levels of cell surface DLL3 and with DLL3 protein and RNA expression levels assessed by immunoblotting and RNAseq. Rova-T showed IC50 values from 0.026-47.25 nM, whereas PBD alone showed IC50 values from 0.003-0.345 nM. Tumors that expressed high DLL3 levels did not necessarily show a higher sensitivity to Rova-T ex vivo. In vivo, Rova-T induced significant differences in EFS distribution compared to control in 3 of 3 PDX models treated with 0.6 mg/kg and in 1 of 2 PDX models dosed at 0.1 and 0.3 mg/kg. All 3 PDX models showed a >2-fold extension in median EFS for treated compared to control animals at 0.6 mg/kg. Two PDXs, COG-N-452x and Felix-PDX, had a minimum relative tumor volume <0.2 to 0.6 mg/kg, and achieved complete and partial responses, respectively. Among the PDXs tested, DLL3 expression was lowest in COG-N-452x; this model showed superior in vivo sensitivity to Rova-T compared with the other 2 models which had higher DLL3 expression and also higher IC50 values for PBD. Conclusions: These data suggest that targeting DLL3 using an ADC approach in NB could be an active therapeutic strategy. Studies are ongoing to further explore biomarkers of response as DLL3 expression alone may be insufficient for NB. Citation Format: Renata Sano, Kateryna Krytska, Matthew Tsang, Stephen W. Erickson, Beverly A. Teicher, Laura Saunders, Ryan T. Jones, Malcom A. Smith, John M. Maris, Yael P. Mosse. Pediatric Preclinical Testing Consortium evaluation of a DLL3-targeted antibody drug conjugate rovalpituzumab tesirine, in neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-136.

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

Initial testing (stage 1) of BAL101553, a novel tubulin binding agent, by the pediatric preclinical testing program.

BAL101553 is a highly water soluble prodrug of BAL27862 that arrests tumor cell proliferation and induces cell death in cancer cells through disruption of the microtubule network. In vitro BAL27862 demonstrated potent activity, with the median relative IC50 (rIC50 ) of 13.8 nM (range 5.4-25.2 nM). The in vitro activity of BAL27862 against the PPTP cell lines is distinctive from that previously described for vincristine. BAL101553 induced significant differences in EFS distribution compared to control in 16 of 30 (53%) solid tumor xenografts and in two of four (67%) of the evaluable ALL xenografts. No objective responses were observed.

Initial testing (stage 1) of the PARP inhibitor BMN 673 by the pediatric preclinical testing program: PALB2 mutation predicts exceptional in vivo response to BMN 673.

BMN 673 is a potent inhibitor of poly-ADP ribose polymerase (PARP) that is in clinical testing with a primary focus on BRCA-mutated cancers. BMN 673 is active both through inhibiting PARP catalytic activity and by tightly trapping PARP to DNA at sites of single strand breaks. BMN 673 was tested in vitro at concentrations ranging from 0.1 nM to 1 μM and in vivo at a daily dose of 0.33 mg/kg administered orally twice daily (Mon-Fri) and once daily on weekends (solid tumors) for 28 days. The median relative IC50 (rIC50 ) concentration against the PPTP cell lines was 25.8 nM. The median rIC50 for the Ewing cell lines was lower than for the remaining cell lines (6.4 vs. 31.1 nM, respectively). In vivo BMN 673 induced statistically significant differences in EFS distribution in 17/43 (39.5%) xenograft models. Three objective regressions were observed: a complete response (CR) in a medulloblastoma line (BT-45), a maintained CR in a Wilms tumor line (KT-10), and a maintained CR in an ependymoma line (BT-41). BMN 673 maintained its high level of activity against KT-10 with a threefold reduction in dose. KT-10 possesses a truncating mutation in PALB2 analogous to PALB2 mutations associated with hereditary breast and ovarian cancer that abrogate homologous recombination (HR) repair. The PPTP results suggest that single agent BMN 673 may have limited clinical activity against pediatric cancers. Single agent activity is more likely for patients whose tumors have defects in HR repair.

Initial testing (stage 1) of the notch inhibitor PF‐03084014, by the pediatric preclinical testing program

PF-03084014, a γ-secretase inhibitor, was tested against the PPTP in vitro cell line panel (1.0 nM to 10 μM) and against the in vivo xenograft panels (administered orally twice daily on Days 1-7 and 15-21). PF-03084014 demonstrated limited in vitro activity, with no cell line achieving ≥50% inhibition. PF-03084014 induced significant differences in EFS distribution in 14 of 35 (40%) solid tumor xenografts, and 1 of 9 ALL xenografts (which lacked a NOTCH1 mutation), but objective responses were not observed. PF-03084014 demonstrated limited single agent activity in vitro and in vivo against the pediatric preclinical models studied.

Abstract C207: Pediatric Preclinical Testing Program (PPTP) stage 1 evaluation of Glembatumumab Vedotin.

Abstract Introduction: Glembatumumab vedotin is an antibody-auristatin conjugate that targets cells expressing the transmembrane glycoprotein NMB (GPNMB, also known as osteoactivin, dc-HIL, and HGFIN). Because of the restrictive expression of GPNMB, testing was limited to the PPTP osteosarcoma (OS) xenograft panel as well as two non-GPNMB expressing rhabdomyosarcoma (RMS) xenografts. Methods: Glembatumumab vedotin was administered intravenously in 0.9% saline at a dose of 2.5 mg/kg using a weekly x 3 schedule. GPNMB was assessed at the RNA level (Agilent SurePrint3) and by IHC. Results: All of the six OS xenografts against which glembatumumab vedotin was tested expressed GPNMB at the RNA level as evaluated using Agilent gene expression arrays. By contrast, none of the RMS xenografts expressed GPNMB at the RNA level. Most OS xenografts showed GPNMB expression detectable by IHC (6/7), while none of the RMS xenografts showed tumor staining for GPNMB (0/6). Glembatumumab vedotin was well tolerated, and all 8 tested xenograft models were considered evaluable for efficacy. For the OS xenografts, glembatumumab vedotin induced significant differences in EFS distribution compared to control in each of the 6 models studied. Three of 6 OS xenografts showed maintained complete responses (MCR) to glembatumumab vedotin. Two other xenografts showed progressive disease with growth delay (PD2), with one of these showing near stable disease during the 6 weeks of treatment and observation. The final OS xenograft showed progressive disease with no growth delay (PD1). As expected, the two RMS xenografts showed limited responses to glembatumumab vedotin (PD1 and PD2), although one did show significant tumor growth inhibition with an EFS T/C value of 2.6. Two of the OS xenografts with MCR showed the highest GPNMB expression at the RNA level. Conversely, the xenograft with the lowest GPNMB gene expression had the poorest response to glembatumumab vedotin. For GPNMB protein expression assessed by IHC, 2 of the xenografts with MCR showed the highest expression level and highest percent tumor expression, but the other responding line showed GPNMB expression on only 5% of tumor cells, despite exhibiting one of the highest expression levels of GPNMB by mRNA. The two OS xenograft lines least responsive to glembatumumab vedotin were also the least responsive OS xenografts to vincristine and eribulin. Conclusions: Glembatumumab vedotin showed high-level activity against 3 of 6 OS xenografts. Response of OS xenografts to glembatumumab vedotin may be related to both GPNMB expression and to the intrinsic susceptibility of xenografts to tubulin inhibitors. RMS xenografts showed PD1 or PD2 responses to glembatumumab vedotin despite having known sensitivity to tubulin-binding agents such as eribulin. Glembatumumab vedotin is the first antibody-drug conjugate with documented in vivo activity against OS. (Supported by NO1-CM-42216) Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C207. Citation Format: Richard Gorlick, Anders Kolb, Jainrong Wu, Raushan Kurmasheva, Thomas Hawthorne, Peter Houghton, Malcolm Smith. Pediatric Preclinical Testing Program (PPTP) stage 1 evaluation of Glembatumumab Vedotin. [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 C207.

Initial testing (stage 1) of the topoisomerase II inhibitor pixantrone, by the pediatric preclinical testing program

Pixantrone, a novel aza-anthracenedione with cytotoxic activity, was tested against the PPTP in vitro panel (3.0 nM to 30.0 μM) and against a limited panel of PPTP Wilms tumors and sarcomas (7.5 mg/kg) administered intravenously using an every 4 day × 3 schedule. In vitro pixantrone showed a median relative IC50 value of 54 nM (range <3 nM to 1.03 μM). In vivo pixantrone induced significant differences in EFS distribution compared to controls in two of eight solid tumor xenografts at dose levels relevant to human drug exposure. A complete response was observed for one Wilms tumor xenograft.

Initial testing (Stage 1) of TAK-701, a humanized hepatocyte growth factor binding antibody, by the pediatric preclinical testing program

TAK-701 is a humanized antibody that binds hepatocyte growth factor (HGF), thus suppressing c-Met transduced signaling and c-Met dependent proliferation and migration of tumor cells. Six childhood solid tumor xenografts were selected for evaluating TAK-701 based on immunochemical detection of HGF/c-Met autocrine signaling [i.e., pMet(Tyr1349) and HGF positive]. TAK-701 was tested using a dose of 30 mg/kg administered by the intraperitoneal (IP) route twice weekly for 4 weeks. TAK-701 did not induce significant differences in EFS distribution in treated tumors compared to control tumors. Objective responses were not observed in any of the tested solid tumor xenografts.

Initial testing (stage 1) of the histone deacetylase inhibitor, quisinostat (JNJ-26481585), by the Pediatric Preclinical Testing Program

Quisinostat (JNJ-26481585) is a second-generation pyrimidyl-hydroxamic acid histone deacetylase (HDAC) inhibitor with high cellular potency towards Class I and II HDACs. Quisinostat was selected for clinical development as it showed prolonged pharmacodynamic effects in vivo and demonstrated improved single agent antitumoral efficacy compared to other analogs. Quisinostat was tested against the PPTP in vitro panel at concentrations ranging from 1.0 nM to 10 μM and was tested against the PPTP in vivo panels at a dose of 5 mg/kg (solid tumors) or 2.5 mg/kg (ALL models) administered intraperitoneally daily × 21. In vitro quisinostat demonstrated potent cytotoxic activity, with T/C% values approaching 0% for all of the cell lines at the highest concentration tested. The median relative IC50 value for the PPTP cell lines was 2.2 nM (range <1-19 nM). quisinostat induced significant differences in EFS distribution compared to control in 21 of 33 (64%) of the evaluable solid tumor xenografts and in 4 of 8 (50%) of the evaluable ALL xenografts. An objective response was observed in 1 of 33 solid tumor xenografts while for the ALL panel, two xenografts achieved complete response (CR) or maintained CR, and a third ALL xenograft achieved stable disease. Quisinostat demonstrated broad activity in vitro, and retarded growth in the majority of solid tumor xenografts studied. The most consistent in vivo activity signals observed were for the glioblastoma xenografts and T-cell ALL xenografts.

Abstract 2758: Pediatric preclinical testing program (PPTP) stage 1 evaluation of MLN0128, a potent TOR kinase inhibitor.

Abstract Introduction: The PI3K-TORC1 pathway links extracellular (growth factors) and intracellular (nutrient sensing) to cell cycle progression and proliferation. This pathway is dysregulated in many adult cancers, but less is known regarding childhood malignancies. MLN0128 is a novel orally-available small molecule inhibitor of both the TORC1 and TORC2 complexes, key components of the PI3K/mTOR signaling pathway. Methods: MLN0128 was evaluated against the 23 cell lines of the PPTP in vitro panel using 96 hour exposure at concentrations from 0.1 nM to 1.0 μM. MLN0128 was tested against the PPTP solid tumor xenografts (SCID mice) and acute lymphoblastic leukemia (ALL; NOD-SCID mice) panels using a dose of 1 mg/kg administered by the P.O. route daily for 21 days. Results: In vitro the median relative IC50 value for the PPTP cell lines was 19 nM, with a range from 2 nM to 102 nM. There was a trend for lower median rIC50 values for the rhabdomyosarcoma and Ewing sarcoma cell line panels (8 nM and 5 nM, respectively). The median rIC50 value for the ALL cell lines (68 nM) was significantly greater than that for the non-ALL cell lines. In vivo MLN0128 was well tolerated, with only a 1.4% toxicity rate in the treated groups, compared to a 0.3% toxicity rate in control animals. All 38 tested xenograft models were considered evaluable for efficacy. MLN0128 induced significant differences in EFS distribution compared to control in 24 of 31 (77%) of the evaluable solid tumor xenografts, but did not induce significant differences in EFS distribution in any of the 7 evaluable ALL xenografts. MLN0128 induced tumor growth inhibition meeting criteria for intermediate EFS T/C activity (EFS T/C &amp;gt; 2) in 6 of 30 (20%) evaluable solid tumor xenografts. Intermediate activity for the EFS T/C metric occurred in the rhabdoid tumor panel (2 of 3) and in single xenografts in four other panels. Objective responses were not observed for the solid tumor or for the ALL xenograft panels. Pharmacodynamic studies to determine TOR inhibition are planned. Conclusions: The activity observed for MLN0128 against the PPTP preclinical models is similar to that previously reported by the PPTP for another TOR kinase inhibitor (Houghton PJ, et al. Pediatr Blood Cancer. 58:191-9, 2012). When combined with PPTP results reported for PI3K and AKT inhibitors (Reynolds CP, et al. Pediatr Blood Cancer. 2012. Epub 2012/09/25 and Gorlick R, et al. Pediatr Blood Cancer. 59:518-24, 2012), the available data suggest that kinase inhibitors targeting the PI3K pathway produce limited single agent activity for the tumor types represented by the PPTP models. (Supported by award NO1-CM-42216 from the NCI). Citation Format: Peter Houghton, Min H. Kang, Patrick Reynolds, Richard Lock, Hernan Carol, Richard Gorlick, Anders Kolb, John Maris, Stephen Keir, Catherine Billups, Raushan Kurmasheva, Malcolm Smith. Pediatric preclinical testing program (PPTP) stage 1 evaluation of MLN0128, a potent TOR kinase inhibitor. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2758. doi:10.1158/1538-7445.AM2013-2758

Abstract LB-354: Pediatric Preclinical Testing Program (PPTP) stage 1 evaluation of the XPO1/CRM1 inhibitor KPT-330.

Abstract Introduction: KPT-330 is an oral Selective Inhibitor of Nuclear Export (SINE) that binds covalently to XPO1 at Cys528 resulting in its irreversible inactivation. The nuclear export of over 200 proteins with specific nuclear export sequences (NES) is mediated via XPO1. Amongst the client proteins are many tumor suppressor and growth regulatory proteins (e.g., FOXO, IκB, pRb, p53, p73, p21, and p27). Methods: KPT-330 was tested against the PPTP's in vitro cell line panel at concentrations ranging from 1.0 nM to 10.0 μM using the PPTP's standard 96 hour exposure period. It was tested against the PPTP solid tumor xenografts using a dose of 10 mg/kg administered by the oral route thrice weekly (M-W-F) for 4 weeks with a total treatment/observation period of 6 weeks. Results: The median relative IC50 (rIC50) value for the PPTP cell lines was 125 nM, with a range from 13 nM to greater than 10 μM. There were no significant differences in rIC50 values by histotype, although there was a trend for greater sensitivity for the Ewing sarcoma cell lines (median rIC50 = 57 nM) and lesser sensitvity for the neuroblastoma cell lines (median rIC50 = 235 nM). Most cell lines showed Relative I/O% values between -75% and -100%, consistent with a prominent cytotoxic effect for KPT-330. KPT-330 was well tolerated in vivo. It induced significant differences in EFS distribution compared to control in 29 of 37 (78%) solid tumor xenografts and in 5 of 8 (63%) ALL xenografts. For those xenografts with a significant difference in EFS distribution between treated and control groups, an EFS T/C value of greater than 2.0 indicates a substantial agent effect in slowing tumor growth. KPT-330 induced this level of effect in 11 of 32 (34%) solid tumor xenografts, most frequently for the Wilms tumor (2 of 3) and the Ewing sarcoma (4 of 5) panels. Objective responses were observed in 3 of 38 (4%) solid tumor xenografts, including a maintained complete response (MCR) for a Wilms tumor xenograft, a CR for a medulloblastoma xenograft, and a CR for a slow-growing ependymoma xenograft. For the ALL panel, 2 of 8 (25%) xenografts achieved either CR (ALL-8, T-cell ALL) or MCR (ALL-19, B-precursor ALL). Conclusions: KPT-330 shows potent in vitro activity against many PPTP cell lines, consistent with the activation of multiple tumor suppressor proteins across diverse tumor genotypes. KPT-330 shows tumor regressing activity against selected PPTP solid tumor and ALL xenografts, and shows tumor growth inhibition for a larger number of models. Defining the relationship between KPT-330 systemic exposure in mice and humans will be important in assessing the clinical relevance of the PPTP in vivo results. Planned PD testing may identify biomarkers associated with response of pediatric preclinical models to KPT-330. KPT-330 is in phase 1 clinical trials in adults with advanced solid or hematological malignancies (NCT01607905 and NCT01607892). (Supported by NCI NO1-CM-42216) Citation Format: Peter Houghton, Min Kang, Patrick Reynolds, Richard Gorlick, Anders Kolb, John Maris, Stephen Keir, Hernan Carol, Richard Lock, Catherine Billups, Raushan Kurmasheva, Yosef Landesman, Sharon Shacham, Michael Kauffman, Malcolm A. Smith. Pediatric Preclinical Testing Program (PPTP) stage 1 evaluation of the XPO1/CRM1 inhibitor KPT-330. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-354. doi:10.1158/1538-7445.AM2013-LB-354

Abstract LB-353: Pediatric Preclinical Testing Program (PPTP) stage 1 evaluation of cabozantinib.

Abstract Introduction: Cabozantinib is an oral, small-molecule inhibitor of MET, VEGFR2, and RET, and it also inhibits AXL, KIT and TIE-2. These targets are known to play important roles in tumor cell proliferation and/or angiogenesis. Cabozantinib is FDA-approved for the treatment of patients with progressive metastatic medullary thyroid cancer. Methods: Cabozantinib was tested against the PPTP's in vitro cell line panel at concentrations ranging from 1.0 nM to 10.0 μM using the PPTP's standard 96 hour exposure period. It was tested against the PPTP solid tumor xenografts using a dose of 30 mg/kg administered by the oral route daily for 3 or 4 weeks with a total treatment/observation period of 6 weeks. Results: The median relative IC50 value for the PPTP cell lines was 8.1μM, with a range from 0.034 μM to greater than 10.0 μM and with only 3 cell lines showing IC50 values less than 1 μM. The most sensitive cell line, Kasumi-1, is an AML cell line that is known to have an activating KIT mutation. Cabozantinib was generally well tolerated in vivo, with only a 1.8% toxicity rate in treated animals. Thirty-four of 34 tested xenograft models were considered evaluable for efficacy. Cabozantinib induced significant differences in EFS distribution compared to control in 31 of 34 (91%) of the evaluable solid tumor xenografts. Cabozantinib induced tumor growth inhibition meeting criteria for intermediate EFS T/C activity (EFS T/C greater than 2) in 22 of 31 (71%) evaluable solid tumor xenografts. Intermediate activity for the EFS T/C metric was observed across all tumor panels evaluated. Partial responses were observed for two of the solid tumor xenografts, the rhabdoid tumor xenograft BT-29 and the slow-growing ependymoma xenograft BT-41. Tumor growth control was most pronounced for the alveolar rhabdomyosarcoma xenografts (ARMS), with 4 of 5 ARMS lines showing less than 20% increase in tumor volume during the 21 days of treatment. Two of 3 Wilms tumor lines (KT-11 and KT-13) also showed complete tumor growth control at Day 21, as did the medulloblastoma xenograft (BT-28) that was studied. Conclusions: The in vitro and in vivo pattern of activity observed for cabozantinib against the PPTP preclinical models is similar to that of agents previously studied by the PPTP that inhibit angiogenesis (e.g., sunitinib, cediranib, and sorafenib). The absence of in vitro activity for most solid tumor cell lines at concentrations associated with specific kinase inhibition together with widespread in vivo tumor growth inhibition and infrequent tumor regression suggest that for most pediatric cancers the in vivo effects of cabozantinib will primarily result from indirect effects on tumor growth (e.g., inhibition of VEGFR2-mediated angiogenesis). Exceptions would be those pediatric cancers with activating genomic alterations in kinases specifically inhibited by cabozantinib (e.g., medullary thyroid cancer with RET mutations). (Supported by NO1-CM-42216 from the NCI). Citation Format: Malcolm Smith, Min Kang, Patrick Reynolds, Richard Gorlick, Anders Kolb, John Maris, Stephen Keir, Catherine Billups, Raushan Kurmasheva, Peter Houghton. Pediatric Preclinical Testing Program (PPTP) stage 1 evaluation of cabozantinib. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-353. doi:10.1158/1538-7445.AM2013-LB-353

Abstract 2755: Pediatric Preclinical Testing Program (PPTP) stage 1 evaluation of NSC750854, a sulfamated purine analog with a distinctive anticancer activity profile.

Abstract Introduction: NSC750854 [3,4-dihydroxy-5-purin-9-yloxolan-2-yl)methyl sulfamate] is a purine analog with an activity profile substantially different from that of other anticancer purines. Unlike purine analogs used as anticancer agents, it does not have any substituents on the purine base. Because of the 5’-sulfamate on its sugar component, it cannot be phosphorylated. Initial mechanistic studies have shown that it does not interact with the RNA/DNA pathways typically affected by purine analogs, and further work on target identification is underway. Methods: NSC750854 was tested against the PPTP's in vitro cell line panel at concentrations ranging from 1.0 nM to 10.0 μM using the PPTP's standard 96 hour exposure period. It was tested against PPTP solid tumor xenografts using a dose of 5 mg/kg administered by the intraperitoneal (IP) route daily for 5 days repeated at day 15. Results: The median relative IC50 (rIC50) value for the PPTP cell lines was 32 nM, with a range from 11 nM (MOLT-4) to 124 μM (CHLA-258). NSC750854 demonstrated consistent cytotoxicity across all of the PPTP cell lines, with Relative In/Out% values near -100% for each of the cell lines. The acute lymphoblastic leukemia (ALL) cell lines were more sensitive to NSC750854 than non-ALL cell lines. NSC750854 showed no dependence on TP53 for its cytotoxic effect, and TP53 mutated cell lines had nominally lower rIC50 values compared to wildtype cell lines. NSC750854 was well tolerated in vivo, with only a 1.7% toxicity rate in treated animals, similar to that observed for control animals (0.7%). NSC750854 induced significant differences in EFS distribution compared to control in 26 of 30 (87%) of the evaluable solid tumor xenografts. NSC750854 induced tumor growth inhibition meeting criteria for intermediate or high EFS T/C activity (requiring EFS T/C &amp;gt; 2) in 14 of 29 (48%) evaluable solid tumor xenografts. Objective responses were observed in 11 of 30 xenografts. Responses were observed for multiple histotypes, including rhabdoid tumors, Wilms tumor, Ewing sarcoma, rhabdomyosarcoma, glioblastoma, and neuroblastoma. While some of the xenografts that were responsive to NSC750854 have also shown responsiveness to other cytotoxic agents (e.g., KT-10, KT-16, and Rh10 and to a lesser extent D456 and D645), other responsive models have shown limited sensitivity to most agents against which they've been tested (e.g., BT-29 and CHLA-79). Conclusions: NSC75084 shows high activity at a well-tolerated dose against multiple pediatric solid tumor xenografts, including activity against highly resistant models. Data mining activities are ongoing to generate hypotheses regarding molecular characteristics associated with responsiveness to NSC750854. (Supported by award NO1-CM-42216 from the NCI). Citation Format: Malcolm Smith, Min Kang, Patrick Reynolds, Richard Gorlick, Anders Kolb, John Maris, Stephen Keir, Catherine Billups, Raushan Kurmasheva, Peter Houghton, Jerry Collins. Pediatric Preclinical Testing Program (PPTP) stage 1 evaluation of NSC750854, a sulfamated purine analog with a distinctive anticancer activity profile. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2755. doi:10.1158/1538-7445.AM2013-2755

Initial testing (stage 1) of ganetespib, an Hsp90 inhibitor, by the pediatric preclinical testing program

Ganetespib, an Hsp90 inhibitor, was tested against the PPTP in vitro cell line panel and selected xenografts in vivo, including JAK2- and BRAF-mutated models. Ganetespib demonstrated potent in vitro cytotoxic activity (median rIC50 8.8 nM, range 4.4-27.1 nM). In vivo, ganetespib induced significant differences in EFS distribution for 4 of 11 xenografts. Intermediate activity (EFS T/C > 2) was noted only for the MV4;11 xenograft, and there were no objective responses. Administered as single agents, Hsp90 inhibitors examined by the PPTP have shown limited evidence for a therapeutic window against both solid tumor and leukemia pediatric preclinical models.

Initial testing (stage 1) of the phosphatidylinositol 3′ kinase inhibitor, SAR245408 (XL147) by the pediatric preclinical testing program

Activation of the PI3 kinase pathway occurs frequently in many adult cancers and is implicated in tumor cell proliferation, survival, and resistance to chemotherapy and radiotherapy. However, less is known regarding the relevance of this pathway in pediatric cancers. Here we have evaluated SAR245408, a novel small molecule PI3K inhibitor, against childhood cancer cell lines and xenografts. SAR245408 was tested against the PPTP in vitro cell line panel at concentrations from 10 to 100 µM and against the PPTP in vivo xenograft panels at a dose of 100 mg/kg administered orally daily × 14. In vitro SAR245408 demonstrated cytotoxic activity, with a median relative IC50 value of 10.9 µM (range 2.7-24.5 µM). SAR245408 was well tolerated in vivo, and all 44 tested xenograft models were evaluable for efficacy. SAR245408 induced significant differences in EFS distribution compared to control in 29 of 37 (79%) of solid tumor xenografts and in two of seven (29%) ALL xenografts. SAR245408 induced tumor growth inhibition meeting criteria for intermediate EFS T/C activity (EFS T/C > 2) in 4 of 37 (11%) solid tumor xenografts. Intermediate EFS T/C activity was also observed for two of seven (29%) evaluable ALL xenografts. Objective responses were not observed for solid tumor or for ALL xenografts. Under the conditions evaluated in this study, SAR245408 achieved modest single-agent activity against most PPTP preclinical models. Further exploration of SAR245408 in combination with standard agents or with other signaling inhibitors could be considered.

Abstract LB-318: Pediatric Preclinical Testing Program (PPTP) evaluation of the JAK inhibitor AZD1480

Abstract Background: AZD1480 is a potent, competitive small molecule inhibitor of JAK1/2 kinase that has entered clinical evaluation. JAK inhibition is of particular pediatric interest given the activating JAK1/2 mutations observed in a subset of pediatric ALL cases. The activity of AZD1480 was evaluated against the PPTP's in vitro and in vivo panels. Methods: AZD1480 (provided by AstraZeneca) was tested in vitro at concentrations from 1.0 nM to 10.0 µM. It was evaluated against solid tumor xenografts at 60 mg/kg administered by oral gavage daily x 5 for 3 weeks, with a total treatment and observation period of 6 weeks. For the ALL panel (using NOD-SCID mice), the maximum tolerated dose was lower, and a twice daily schedule was utilized: 10 mg/kg BID (with a single daily dose of 15 mg/kg on weekends). Standard PPTP measures of in vivo antitumor activity were employed to assess response to AZD1480. Results: The median relative IC50 (rIC50) for AZD1480 against the PPTP cell lines was 1.5 µM, with a range from 0.3 µM to 5.9 µM. AZD1480 induced significant differences in EFS distribution compared to control in 25 of 27 (93%) evaluable solid tumor xenografts. AZD1480 induced tumor growth inhibition meeting criteria for intermediate or high EFS T/C activity in 11 of 26 (42%) solid tumor xenografts evaluable for this measure. Both Wilms tumor xenografts tested showed EFS T/C &amp;gt; 2, as did 2 of 4 GBM xenografts and 2 of 4 neuroblastoma xenografts. An objective response was observed for 1 solid tumor xenograft, a Wilms tumor xenograft KT-10 that achieved a maintained complete response (MCR). Many solid tumor xenografts show phospho-STAT3 expression, but this marker showed no discernible relationship with response to AZD1480. For the ALL panel, 5 JAK mutated xenografts (3 JAK2 and 2 JAK1) were selected for testing to determine whether AZD1480 shows high activity in models in which the JAK-STAT pathway is activated by mutation. Additionally, 4 non-JAK mutated xenografts were evaluated. Models with JAK mutations show phospho-STAT5 as evidence of JAK-STAT signaling. However, the only ALL xenograft with EFS T/C &amp;gt; 2 was a JAK2 mutant (R867Q) xenograft, TGT-20, and no models showed objective responses (PR or CR). Conclusions: AZD1480 showed tumor growth inhibitory activity against most of the solid tumor xenografts, and induced an objective response (an MCR) in a single Wilms tumor xenograft. Genomic sequencing is being undertaken to determine whether this xenograft has a genomic alteration(s) in a JAK family kinase or another kinase that may explain its favorable response. No objective responses were noted for the ALL panel, even among xenografts with JAK mutations. Our results suggest that inhibition of JAK signaling alone may not be sufficient for clinical activity against JAK-mutated ALL. (Supported by NCI NO1CM42216) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-318. doi:1538-7445.AM2012-LB-318

Evaluation of arsenic trioxide by the pediatric preclinical testing program with a focus on Ewing sarcoma

Arsenic trioxide was tested against the PPTP in vitro panel (1.0 nM to 10 µM) and against the PPTP Ewing sarcoma in vivo panel administered intraperitoneally at a dose of 2.5 mg/kg daily × 5 per week for a planned treatment duration of 3 weeks. Arsenic trioxide showed a median relative IC(50) value of 0.9 µM, with Ewing sarcoma cell lines having IC(50) values similar to those of the remaining PPTP cell lines. Arsenic trioxide did not induce significant differences in EFS distribution compared to control in any of the Ewing sarcoma xenografts studied, and no objective responses were observed.

Testing of the Akt/PKB inhibitor MK‐2206 by the pediatric preclinical testing program

MK-2206 is a small molecule allosteric inhibitor of Akt/PKB that is undergoing clinical trials for treatment of cancer. MK-2206 was tested against the PPTP in vitro panel using a 96-hour exposure (1.0 nM-10 µM), and in vivo using thrice weekly dosing for a planned 4 weeks at its maximum tolerated dose (MTD) of 180 mg/kg. In vitro, the median relative IC(50) value for MK-2206 was 2.2 µM. Four cell lines with IC(50) values < 200 nM included two ALL cell lines (COG-LL-317 and RS4;11), an AML cell line with an activating KIT mutation (Kasumi-1), and a Ewing sarcoma cell line (CHLA-10). In vivo, MK-2206 induced significant differences in EFS distribution compared to control in 12 of 29 (41%) of the evaluable solid tumor xenografts and in 2 of 8 (25%) of the evaluable ALL xenografts. Significant differences in EFS distribution were most frequently noted in the osteosarcoma panel (6 of 6). A single solid tumor xenograft (OS-31) had a greater than twofold increase in time to event compared to control animals, with all other solid tumor xenografts showing lesser degrees of tumor growth inhibition. Objective responses were not observed for either the solid tumor or ALL xenografts. MK-2206 showed its most consistent activity in vitro against ALL cell lines and in vivo against osteosarcoma xenografts. However, no objective responses were observed in solid tumor or ALL xenografts. Further preclinical work evaluating MK-2206 in pediatric models in the combination therapy setting may contribute to its pediatric development.

Abstract C106: Pediatric Preclinical Testing Program (PPTP) Stage 1 evaluation of the PI3K inhibitor XL147 (SAR245408).

Abstract Introduction: XL147 is a potent, orally bioavailable inhibitor of the class I PI3K family of lipid kinases with IC50 values in the nanomolar range in biochemical assays. XL147 inhibits phosphorylation of downstream effectors of PI3K in preclinical models as well as in surrogate tissues and tumors in adults with cancer. XL147 is under clinical evaluation for adults with cancer in combination with certain cytotoxic agents as well as with other cell signaling pathway inhibitors (e.g., the MEK inhibitor MSC1936369B). The activity of XL147 was evaluated against the in vitro and in vivo panels of the Pediatric Preclinical testing Program (PPTP). Methods: XL147 was tested against the PPTP's in vitro cell line panel at concentrations ranging from 10.0 nM to 100 μM using the PPTP's standard 96 hour exposure period. For in vivo testing, an XL147 dose of 100 mg/kg administered orally daily for 14 days with a total planned observation period of 6 weeks. Results: XL147 demonstrated cytotoxic activity, with Ymin values approaching 0% for all of the cell lines at the highest concentration tested (100 μM). The median relative IC50 value for the PPTP cell lines was 10.9 μM, with a range from 2.7 μM (CHLA-10) to 24.5 μM (TC-71). There were no significant differences by histotype in median relative IC50 values, though there was a trend for lower values for the rhabdomyosarcoma panel (median IC50 5.6 μM) and higher values for the neuroblastoma panel (median IC50 19.5 μM). XL147 was tested against 31 solid tumor xenografts and 7 acute lymphoblastic leukemia (ALL) xenografts. A dose of 100 mg/kg administered orally daily for 14 days for a total planned observation period of 6 weeks was utilized. XL147 was generally well tolerated, with a &amp;lt;1% toxicity rate in the treated groups, similar to that observed for control animals. XL147 induced significant differences in EFS distribution compared to control in 26 of 30 (87%) of the evaluable solid tumor xenografts and in 2 of 7 (29%) of the evaluable ALL xenografts. XL147 induced tumor growth inhibition meeting criteria for intermediate EFS T/C activity (EFS T/C &amp;gt; 2) in 3 of 29 (10%) evaluable solid tumor xenografts (2 of 6 rhabdomyosarcoma and 1 of 5 neuroblastoma), and intermediate or high EFS T/C activity was observed for 2 of 7 (29%) evaluable ALL xenografts. Basal levels of activation of PI3K signaling, as assessed by phospho-AKT, levels did not correlate with in vivo response to XL147. Expression of PI3K isoforms at the RNA level was evaluated using Affymetrix U133 Plus 2.0 arrays, with the most striking observation being the pattern of expression of PIK3CD, which was virtually restricted to the ALL and lymphoma cell lines and xenografts, as would be expected from the hematopoietic cell specificity of this isoform in normal tissues. Conclusions: Under the conditions evaluated in this study, XL147 achieved modest single-agent activity against the PPTP preclinical models. Available data suggests that genomic activation of PI3K signaling (e.g., through PIK3CA mutation or PTEN deletion) is less common in the pediatric solid tumors setting compared to the adult cancer setting, which may explain the limited single agent activity. Preclinical evaluations of XL147 in combination with particular standard agents and with other pathway signaling inhibitors are planned. 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 C106.

Abstract C105: Pediatric Preclinical Testing Program (PPTP) Stage 1 evaluation of the CD56-targeting antibody-drug conjugate lorvotuzumab mertansine.

Abstract Introduction: Lorvotuzumab mertansine (LM) is a conjugate consisting of the cytotoxic maytansinoid, DM1, covalently linked to the humanized monoclonal antibody lorvotuzumab, which selectively binds to CD56 (NCAM, neural cell adhesion molecule). LM is currently in clinical trials for patients with CD56-positive cancers [e.g., small-cell lung cancer (SCLC), multiple myeloma, and Merkel cell carcinoma]. The activity of LM was evaluated against the in vitro and in vivo panels of the Pediatric Preclinical testing Program (PPTP). Methods: LM and its cytotoxic moiety L-DM1-SMe were tested against the PPTP's in vitro cell line panel at concentrations ranging from 0.01 nM to 0.1 μM using the PPTP's standard 96 hour exposure period. LM was tested against a subset of PPTP solid tumor xenografts at a dose of 15 mg/kg by the intravenous route using a weekly × 3 schedule with a total planned testing and observation period of 6 weeks. Models were selected for testing based on their CD56 expression levels. Results: The relative IC50 (rIC50) for LM and L-DM1-SMe were 35 nM and 2 nM, respectively. The two agents had very distinctive activity patterns, with LM showing its greatest activity against the neuroblastoma panel, and with L-DM1-SMe showing its highest activity against the ALL panel. L-DM1-SMe showed 79-fold greater potency against the ALL panel (median rIC50 1.1 nM) compared to the neuroblastoma panel (median rIC50 84 nM). By contrast, LM showed 1.7-fold greater potency against the neuroblastoma panel (median rIC50 32 nM) compared to the ALL panel (median rIC50 53 nM). The cell lines of the neuroblastoma panel had NCAM (CD56) expression at or above the median expression level for all PPTP xenografts and cell lines, and these cell lines demonstrated corresponding high levels of sensitivity to LM compared to L-DM1-SMe. The converse was true for the ALL cell lines, which had low NCAM (CD56) expression and low sensitivity to LM compared to L-DM1-SMe. In vivo, LM induced significant differences in EFS distribution compared to control in 15 of 18 (83%) of the evaluable solid tumor xenografts, including all 7 neuroblastoma xenografts. Objective responses were observed in 6 of 18 (33%) solid tumor xenografts [2 complete responses (CR) and 4 maintained CR (MCR)]. CR/MCR responses were observed for 3 of 7 neuroblastoma xenografts and 2 of 5 rhabdomyosarcoma xenografts. The other MCR observed was for the Wilms tumor xenograft KT-10. Each of the 6 xenografts achieving CR or MCR responses showed homogeneous staining by IHC for NCAM (CD56) with expression levels of 3 or 3+ (excepting NB-1643 which showed 2–3 level homogeneous staining). However, other xenografts also showed 3 or 3+ homogeneous staining but did not show tumor regression in response to LM treatment. Comparison of LM in vivo activity to that previously described for vincristine showed that neuroblastoma xenografts with data for both agents were more responsive to LM than to vincristine. Conclusions: LM shows target-directed activity in vitro and promising activity in vivo against CD56-expressing childhood cancers. Combination testing with topotecan is planned for the neuroblastoma panel to take advantage of previously reported synergistic interactions for topoisomerase-I inhibitors administered with microtubule-targeting agents (Cancer Chemother Pharmacol 2001:47; 211–21). 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 C105.