Abstract 7041: The CLK/DYRK inhibitor SM09419 shows potent efficacy across a broad panel of pediatric preclinical xenograft models - A report from the Pediatric Preclinical In Vivo Testing Consortium (PIVOT)

Pediatric Preclinical Testing Consortium Evaluation of the Novel Anti-Microtubule Drug E7130 in Xenograft Models of Early T-Cell Precursor Acute Lymphoblastic Leukemia

Pediatric Preclinical Testing Consortium Evaluation of the MCL1 Inhibitor, AMG 176, Against Xenograft Models of Acute Lymphoblastic Leukemia

Introduction: While children with acute lymphoblastic leukemia (ALL) experience close to a 90% likelihood of cure, the outcome for certain high-risk pediatric ALL subtypes remains poor. Spleen Tyrosine Kinase (SYK), a cytosolic nonreceptor tyrosine kinase, is primarily expressed in the hematopoietic lineage and is essential for B-cell receptor signaling. It is associated with malignant transformation, cancer cell proliferation, and is a prominent tyrosine-phosphorylated protein in B-lineage pediatric ALL (Dolai et al, Cancer Res 76:2766-77, 2016). Fms Related Receptor Tyrosine Kinase 3 (FLT3) is a Class III receptor tyrosine kinase that regulates hematopoiesis. While uncommon in ALL, activating FLT3 mutations and internal tandem duplications are associated with poor outcome. TAK-659 is a dual SYK/FLT3 reversible inhibitor currently undergoing clinical trials against B-cell lymphoma, acute myeloid leukemia and solid tumors. Therefore, it was of interest for the Pediatric Preclinical Testing Consortium to test TAK-659 in vivo against its patient-derived xenograft (PDX) models of pediatric ALL. Methods: SYK and FLT3 mRNA expression in ALL PDXs was quantified by RNAseq (https://pedcbioportal.org) and 8 B-lineage pediatric ALL PDXs (3 B-cell precursor, BCP; 4 mixed-lineage leukemia-rearranged, MLLr; one Philadelphia Chromosome-like, Ph-like) were selected for testing. Engraftment was evaluated by enumerating the proportion of human CD45+ cells (%huCD45+) in the peripheral (PB) blood at weekly intervals. TAK-659 was tested at 60 mg/kg by oral gavage daily for 21 days. Events were defined as %huCD45+ ≥ 25%. At Day 28 post-treatment initiation or event (whichever occurred first), 4 mice/group were humanely killed to assess leukemia infiltration in the spleen and bone marrow (BM). Drug efficacy was assessed by event-free survival (EFS) of treated (T) and control (C) groups by T-C, T/C and stringent objective response measures (Houghton et al, Pediatr Blood Cancer 49:928-40, 2007). Results: The in vivo efficacy of TAK-659 was evaluated against B-lineage PDXs as FLT3 and SYK mRNA expression was higher in the PDXs from this lineage compared with PDXs from T-ALL. TAK-659 was well tolerated and significantly prolonged the time to event in 6/8 PDXs (T-C 0-25.5 days, T/C 1.0-2.2). Only one MLLr-ALL obtained an objective response (partial response) with all other models exhibiting progressive disease. The minimum mean %huCD45+ was significantly reduced in 5/8 PDXs in TAK-659 treated mice compared to control. Despite significant reductions in %huCD45+ in the PB for 5/8 PDXs, TAK-659 did not significantly reduce the spleen or BM infiltration of any PDXs. Conclusion: TAK-659 exhibited low to moderate single-agent in vivo activity against pediatric B-ALL PDXs representative of diverse disease subtypes. (Supported by NCI Grants CA199000 and CA199922) Citation Format: Richard B. Lock, Kathryn Evans, Narimanne El-Zein, Eric J. Earley, Stephen W. Erickson, Beverly A. Teicher, Malcolm A. Smith. Pediatric preclinical testing consortium evaluation of the dual SYK/FLT3 inhibitor TAK-659 in xenograft models of pediatric acute lymphoblastic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3039.

Introduction: CD47 is a cell surface marker that interacts with signal regulatory protein alpha (SIRPα) on macrophages and dendritic cells to negatively regulate phagocytosis. This interaction elicits a “don't eat me” signal preventing phagocytosis by immune cells, enabling cell survival. CD47 is expressed on tumor cells, including acute leukemia cells. AO-176 is a highly differentiated monoclonal antibody that targets CD47, exerts potent in vivo activity in mouse xenograft models, has an established safety profile in cynomolgus monkeys, and is being evaluated in clinical trials. Given the broad expression of CD47 in malignant hematopoietic cells, it was of interest for the Pediatric Preclinical Testing Consortium to evaluate AO-176 in vivo against patient-derived xenograft (PDX) models of pediatric ALL. Methods: CD47 mRNA and protein expression were quantified by RNAseq (https://pedcbioportal.org) and flow cytometry (relative fluorescence intensity, RFI), respectively. AO-176 was tested in vivo against 3 T-ALL (ALL-8, ALL-30, ALL-121) and one early T-cell precursor (ETP) ALL (ETP-2) PDXs in NSG mice at 25 mg/kg intraperitoneally once per week for 4 weeks. Engraftment was evaluated by enumerating the proportion of human CD45+ cells in the peripheral blood (%huCD45+) at weekly intervals. Events were defined as the %huCD45+ ≥ 25%. At Day 28 post-treatment initiation or event (whichever occurred first), 3 mice/group were humanely killed to assess leukemia infiltration of hematolymphoid organs. Drug efficacy was assessed by event-free survival (EFS) of treated (T) and control (C) groups by T-C, T/C, and stringent objective response measures (Houghton et al, Pediatr Blood Cancer 49:928-40, 2007). Results: CD47 mRNA expression was significantly higher in a panel of T-lineage ALL PDXs (n=25; mean + SD FPKM, 149 + 57.9) compared with B-ALL PDXs (n=65; 65.3 + 18.7 FPKM) (P<0.0001, unpaired t-test). Cell surface CD47 expression in a subset of 8 PDXs (6 T-ALL, 2 ETP-ALL) was a median of 40.2 (RFI range 17.9-81.9). AO-176 was well tolerated in NSG mice and significantly (P<0.006) delayed the progression of 3/4 PDXs tested (3 T-ALL, one ETP-ALL) (T-C, 12.7-56.9 days; T/C 3.4-4.9) and one PDX elicited an objective response (Partial Response). AO-176 was least effective against the lowest CD47 expressing PDX. AO-176 significantly (P<0.005) decreased human leukemia infiltration of murine spleens, but not bone marrow, in 3/4 PDXs. Conclusion: Despite being tested in highly immune-deficient mice, the naked antibody AO-176 exhibited significant single-agent in vivo anti-leukemic activity against pediatric T-lineage ALL PDXs. The significant decrease in spleen infiltration elicited by AO-176 in 3/4 PDXs suggests on-target activity consistent with the known mechanism of action of CD47 targeting agents. (Supported by NCI Grants CA199000 and CA199922) Citation Format: Richard B. Lock, Kathryn Evans, Narimanne El-Zein, Eric J. Earley, Stephen W. Erickson, Beverly A. Teicher, Victoria Sung, Malcolm A. Smith. The differentiated CD47 monoclonal antibody AO-176 exhibits significant in vivo activity against xenograft models of pediatric acute lymphoblastic leukemia (ALL) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB171.

Introduction: While overall survival for pediatric acute lymphoblastic leukemia (ALL) now approaches 90%, new treatment options are urgently required for certain high-risk subtypes including BCR-ABL1 or TCF3-HLF ALL, and relapsed T-cell ALL (T-ALL). CXCR4 (CD184) is the most frequently over-expressed chemokine receptor in hematological malignancies, including ALL. Binding of CXCL12 to its cognate receptor CXCR4 promotes tumor progression, metastasis and cell survival through multiple signaling pathways, including ERK1/2, RAS, p38 MAPK, PLC/MAPK, SAPK/JNK and cancer stem cell regulation. X4-136 (X4 Pharmaceuticals) is a second-generation potent, orally available, allosteric CXCR4 inhibitor under clinical evaluation. Therefore, it was of interest for the Pediatric Preclinical Testing Consortium to evaluate the in vivo activity of X4-136 in combination with established drugs against patient-derived xenograft (PDX) models of aggressive pediatric ALL. Methods: CXCR4 expression was quantified by RNAseq (https://pedcbioportal.org). Specific antibody binding capacity (sABC) was assessed using the CELLQUANT Calibrator. X4-136 was tested in vivo in NSG mice against 3 ALL PDXs (ALL-4, BCR-ABL1; ALL-7, TCF3-HLF; ALL-31, relapsed T-ALL) at 100 mg/kg/day PO for 4 weeks either as a single agent or in combination with an induction-type regimen of vincristine, dexamethasone and L-asparaginase (VXL). Events were defined as the proportion of human CD45+cells in the peripheral blood (%huCD45+) ≥25%. Drug efficacy was assessed by event-free survival (EFS) of treated (T) and control (C) groups by T-C, T/C and stringent objective response criteria (Houghton PJ, et al. Pediatr Blood Cancer, 2007) and leukemia infiltration into the femoral bone marrow on Day 28 post treatment initiation. Results: RNAseq showed significantly higher (P<0.0001) CXCR4 expression in ALL PDXs (n=90) compared with 154 PDXs derived from 9 other pediatric cancer histotypes. Median CXCR4 sABC on 29 ALL PDXs ranged from 0 to 3,796 (median 476). ALL-4 (median sABC 901), ALL-7 (3,796) and ALL-31 (1,483) were selected for in vivo efficacy studies. X4-136 was generally well tolerated, with maximum average animal weight loss of 4.5 to 8.8% for the 3 PDXs. Single-agent X4-136 significantly (P<0.05) delayed the progression of all 3 PDXs (T-C 1.8-16 days, T/C 1.4-2.2) but elicited no objective responses of the disease. In contrast, X4-136 combined with VXL delayed disease progression by 28.4-35.7 days (T/C 3.8-7.9) and elicited objective responses in all 3 PDXs (2 Complete Responses, 1 Maintained Complete Response). Moreover, the X4-136/VXL combination was significantly more effective than X4-136 and VXL alone against ALL-4 (P<0.05) and ALL-31 (P<0.01) but not ALL-7. Finally, the X4-136/VXL combination significantly decreased leukemia bone marrow infiltration compared with X4-136 and VXL alone in ALL-7 and ALL-31 (P<0.0001). Conclusions: The addition of X4-136 to an established anti-leukemia regimen significantly prolongs time to event in preclinical models of aggressive childhood ALL. Our results support further evaluation of X4-136 in combination with established drugs for pediatric ALL. (Supported by NCI Grants CA199000 and CA199922) Citation Format: Richard B Lock, Kathryn Evans, Connor D Jones, Narimanne El-Zein, Stephen W Erickson, Sarah Beaussant-Cohen, E. Lynne Kelley, Beverly A Teicher, Malcolm A Smith. The CXCR4 inhibitor X4-136 enhances the in vivo efficacy of established drugs against preclinical models of aggressive pediatric acute lymphoblastic leukemia [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C004. doi:10.1158/1535-7163.TARG-19-C004

Cytidine analogs target DNA methyltransferases as at least one of their mechanisms of action. The mechanism(s) of action of these compounds are incompletely elucidated and vary. TdCyd and Aza-TdCyd are thio-nucleoside analogs of cytidine which differ by a single nitrogen atom in the cytidine ring. Here the activity in solid and liquid tumor cell lines, and pediatric tumor xenografts is compared. Methods: Cytotoxicity was examined after 2, 3 and 7-day exposure in 10 solid tumor lines and 6 liquid tumor lines. TdCyd was administered (1mg/kg) ip, daily for 5 days for 4 weeks (total 20 doses). Aza-TdCyd was administered (1.5, 1.0 or 0.5 mg/kg) ip, daily for 5 days for 4 weeks (total 20 doses). For solid tumors (sc), events were defined as 4X increase in tumor volume from day 0, for acute lymphoblastic leukemias (ALL) as %hCD45 cells exceeding 25%. The Kaplan-Meier method was used to compare time-to-event across treated and control groups. The objective response categories are progressive disease (PD), stable disease (SD), partial response (PR), complete response (CR), and maintained complete response (MCR) [Pediatr Blood Cancer 2007;49:928-40]. Results: Aza-TdCyd was, in general, a more active antitumor agent than TdCyd. In cell culture, after 7-days exposure Aza-TdCyd was 3-to 4-fold more potent than TdCyd in 10 solid tumor lines (IC50 1.2 vs 3.7 uM) and similarly more potent in 6 liquid tumor lines (0.03 vs 0.11 uM). TdCyd and Aza-TdCyd were tested in Ewing sarcoma, rhabdoid tumor, rhabdomyosarcoma, osteosarcoma, and pediatric acute lymphoblastic leukemia (ALL) patient-derived xenografts. The sarcomas were implanted subcutaneously, and the leukemias were implanted intravenously. At tolerable doses, the anticancer activity of both TdCyd and Aza-TdCyd were modest in the Ewing sarcoma (4) and rhabdomyosarcoma (4) models. The activity of both compounds was also modest in 5 out of 6 osteosarcoma models. However, the median survival of mice bearing the OS-9 osteosarcoma doubled (42 days) upon treatment with TdCyd and increased nearly 4-fold (84 days) in mice treated with Aza-TdCyd. While TdCyd had modest ALL activity, Aza-TdCyd increased median survival from 9.1(2-16) days in controls to >70 in 7/9 ALL models. Aza-TdCyd antileukemic activity was maintained at lower doses of the agent with median survivals of 75.4 days at 1.5mg/kg, 72.7 days at 1.0 mg/kg and 62.0 days at 0.5 mg/kg. Both TdCyd (NCT02423057) and Aza-TdCyd (NCT03366116) are in phase 1 clinical testing. Supported by NCI Grants: CA199222, CA199221, CA199297, CA199288, CA199000 and NCI Contract No. HHSN261200800001E. Citation Format: Beverly A. Teicher, Richard B. Lock, Kathryn Evans, Peter J. Houghton, Raushan T. Kurmasheva, Richard Gorlicki, Steve Erickson, Donn Wishka, Joel Morris, Michael Difilippantonio, Jerry E. Collins, Malcolm A. Smith, James H. Doroshow. Comparison of thio-deoxy-cytidine (TdCyd) and aza-thio-deoxy-cytidine (Aza-TdCyd) in solid and liquid tumor cell lines and PPTC pediatric xenografts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3839.

Introduction: Exportin 1 (XPO1/CRM1) plays a central role in the export of proteins from the nucleus including those with tumor suppressor and growth regulatory activity. Eltanexor (KPT-8602) is a second-generation Selective Inhibitor of Nuclear Export (SINE) that specifically blocks XPO1 cargo interactions and is under clinical evaluation for adults with cancer (NCT02649790). In previous Pediatric Preclinical Testing Consortium (PPTC) studies, eltanexor showed potent in vivo activity against acute lymphoblastic leukemia (ALL) patient-derived xenografts (PDXs). In an effort to understand whether eltanexor exhibited subtype-specific activity in pediatric ALL we carried out a single-mouse trial (SMT) against an intended 90 pediatric ALL PDXs. Methods: Pediatric ALL PDXs, an orthotopic model of the disease, were inoculated into NSG mice in an SMT format (a single mouse inoculated with a single PDX was treated with a single drug). Engraftment and drug responses were assessed by weekly enumeration of the % human leukemic blasts in the peripheral blood (%huCD45+/HLA-ABC+). Treatment commenced when the median %huCD45+/HLA-ABC+ exceeded 1%, and mice received eltanexor at 12.5 mg/kg per oral daily x 5 for 4 weeks. The baseline level of the %huCD45+/HLA-ABC+ in each mouse served as its own control. PDX responses to treatment were assessed by time to event, the maximum decrease in %huCD45+/ABC+ at any point after treatment initiation, and by stringent Objective Response Measures (ORMs) modeled after the clinical setting (Houghton et al, Pediatr Blood Cancer, 2007, 49:928-40). Results: Eltanexor was well tolerated with a mean maximum weight loss of 5.5% across all mice. SMT testing was successful in all 90 pediatric ALL PDXs representative of B-ALL (typical B-ALL, Philadelphia chromosome-positive ALL [Ph+-ALL] and Ph-like ALL), T-ALL (typical T-ALL and early T-cell precursor ALL [ETP-ALL]), and MLL-rearranged ALL (MLLr-ALL). Event-free survival (EFS) of mice ranged from 2.6-137 days, and 61 (68%) of mice experienced EFS that extended beyond the treatment window. Regressions of ≥50% from baseline were observed in 56 (62%) of mice, and these were most pronounced in T-ALL (14/18, 78%) and Ph-like ALL (11/19, 58%). ORMs classified as Complete Response (CR) or Maintained CR (MCR) were elicited in 43 (48%) of PDXs, and again these were more common in T-ALL (12/18, 67%). The ALL subtypes that exhibited the poorest responses to eltanexor were Ph+-ALL and ETP-ALL, although with relatively small sample size (n=3 and 6, respectively). ORMs in the SMT correlated highly with historical conventional drug testing (R=0.94, P<0.001, n=12). Conclusions: Eltanexor exhibits potent single-agent in vivo activity against PDXs derived from a broad range of ALL subtypes including T-ALL and Ph-like ALL and warrants further investigation in pediatric ALL. (Supported by NCI Grants CA199222 & CA199000) Citation Format: Richard B. Lock, Kathryn Evans, Connor D. Jones, Stephen W. Erickson, Beverly A. Teicher, TJ Unger, Yosef Landesman, Malcolm A. Smith. The XPO1 inhibitior, eltanexor, exhibits potent in vivo activity against a broad range of pediatric acute lymphoblastic leukemia subtypes [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 4181.

Targeting the apoptosis pathway with BH3 mimetics such as ABT-263 (navitoclax) and ABT-199 is an appealing strategy for cancer therapy. While the Bcl-2/Bcl-w/Bcl-xL inhibitor navitoclax has shown promising activity in adults with lymphoid malignancies and in preclinical models of pediatric acute lymphoblastic leukemia (ALL), its application in pediatric patients has been limited by Bcl-xL-mediated thrombocytopenia. The specific Bcl-2 inhibitor ABT-199 has shown remarkable efficacy against adult chronic lymphocytic leukemia in recent clinical trials, although its activity against pediatric ALL is yet to be tested. The aim of this study was to evaluate the in vivo efficacy of ABT-199 against a large panel of pediatric ALL patient-derived xenografts (PDXs), and to identify biomarkers predictive of ABT-199 response. Immune-deficient (NOD/SCID or NSG) mice inoculated with PDXs derived from patients with infant ALL harboring rearrangement of the Mixed Lineage Leukemia (MLL) oncogene (infant MLL-ALL, n = 4), B-cell precursor ALL (BCP-ALL, n = 6), BCP-ALL categorized as Ph-like (n = 4), T-cell ALL (T-ALL, n = 4) or early T-cell precursor ALL (ETP-ALL, n = 2) were treated with ABT-199 (100 mg/kg x 21 days, p.o.) or vehicle control. Responses were assessed by time to event measurements or stringent objective response criteria modeled after the clinical setting. ABT-199 significantly delayed the progression of 12/20 PDXs by between 0.4 and 28 days, and elicited objective responses in 6/20 (30%) of PDXs (4 complete responses and 2 partial responses). No objective responses were observed in the T-ALL or ETP-ALL PDXs. By comparison, navitoclax administered on the same dose and schedule as ABT-199 elicited objective responses in 19/31 (61%) of PDXs derived from the same pediatric ALL subtypes. Analysis of basal gene and protein expression revealed that high Bcl-xL and low Bcl-2 expression were significantly associated with in vivo ABT-199 resistance. Moreover, the Bcl-2/Bcl-xL gene (P = 0.03) and protein (P = 0.002) expression ratios were significantly elevated in PDXs that responded to ABT-199 in vivo compared with non-responders. Notably, Mcl-1 expression at the gene or protein level showed no significant correlation with in vivo ABT-199 sensitivity. In conclusion, the inferior objective response rate observed for ABT-199 (30%) compared with navitoclax (61%) indicates that pediatric ALL PDXs are less dependent on Bcl-2 for cell survival compared with adult chronic lymphocytic leukemia. Moreover, Bcl-xL appears to be a more significant ABT-199 resistance factor than Mcl-1 in pediatric ALL. While ABT-199 has the potential to exert significant efficacy in the treatment of aggressive and chemoresistant pediatric ALL, the prospective identification of patients who might benefit from such treatment is of paramount importance. Supported by NCI NO1CM42216. Citation Format: Santi Suryani, Kathryn Evans, Jennifer Richmond, Alissa Robbins, Lauryn Bracken, Raushan Kurmasheva, Peter J. Houghton, Malcolm A. Smith, Richard B. Lock. Evaluation of the Bcl-2 inhibitor ABT-199 in xenograft models of acute lymphoblastic leukemia by 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 3276. doi:10.1158/1538-7445.AM2015-3276

Cisplatin is one of the most widely used drugs for the treatment of solid tumors in adults and children. Here, we report the activity of cisplatin against the PPTP panels of childhood cancer xenografts. Cisplatin was evaluated against 23 cell lines, and 40 xenografts representing brain tumors, neuroblastoma, rhabdoid tumors, sarcoma, Wilms tumor, and acute lymphoblastic leukemia (ALL). The IC(50) concentration in vitro was determined for 96 hr exposure. Solid tumors were grown subcutaneously in immune-deficient mice, and tumor dimensions measured weekly. ALL xenografts were inoculated intravenously and the percent human CD45(+) cells in the peripheral blood determined weekly. The antitumor activity of cisplatin (7 mg/kg administered intraperitoneally on Days 0 and 21) was evaluated using time to event (EFS T/C), tumor growth delay (tumor volume T/C), and objective response measures. The median IC(50) concentration in vitro was 0.87 microM (0.24-4.29 microM), and cisplatin exhibited broad range activity. Cisplatin induced significant differences in EFS distributions compared to controls in 20/28 solid tumors and 4/8 ALL models. Objective responses were observed in 7/28 solid tumor models (25%): partial responses in three rhabdomyosarcomas and one Ewing's sarcoma; complete responses in one rhabdoid tumor and the medulloblastoma; and a maintained complete response in one Wilms tumor. No objective responses were observed in the ALL panel. Cisplatin exhibits significant antitumor activity against a broad range of solid tumor xenograft models and limited activity against ALL xenografts. This preclinical pattern of activity is generally consistent with cisplatin's clinical activity.

Despite the success of current therapies for pediatric acute lymphoblastic leukemia (ALL) more effective treatments are required for the management of high-risk subtypes. Ph-like ALL is a high-risk subtype defined by a gene expression signature similar to that of BCR-ABL1-positive ALL despite the absence of the BCR-ABL1 translocation. Approximately 50% of Ph-like pediatric ALLs harbor mutations in Janus kinases (JAKs). Birinapant is a small molecule SMAC (second mitochondria-derived activator of caspase) mimetic that potently and specifically antagonizes inhibitors of apoptosis proteins (IAPs), resulting in IAP degradation, inactivation of NF-κB survival signaling and tumor necrosis factor (TNF)-dependent apoptosis. The combination of birinapant plus azacitidine is being evaluated in a Phase 2 clinical trial for the treatment of high-risk myeloid dysplastic syndrome. The aim of this study was to evaluate the efficacy of birinapant against patient-derived xenografts (PDXs) of pediatric ALL subtypes. Birinapant (30 mg/kg IP Q3 days × 5) significantly delayed the progression of 17/19 PDXs derived from Ph-like ALL (n = 7), B-cell precursor ALL (BCP-ALL, n = 8), and infant MLL¬-rearranged ALL (MLL-ALL, n = 4) by between 2 and 80 days compared with vehicle-treated controls. Using stringent objective response criteria modeled after the clinical setting, birinapant induced objective responses in 12/19 PDXs, including 7/7 Ph-like ALL (5 complete responses, CRs; 2 maintained CRs, MCRs), which was significantly better than BCP-ALL (4/8; 2 partial responses, PRs; 1 CR; 1 MCR) or infant MLL-ALL (1/4; 1 CR) PDXs (P<0.05). Birinapant induced a CR in a Ph-like PDX at a dose 1/8th (3.8 mg/kg) of its maximum tolerated dose (30 mg/kg). Analysis at 14 days following treatment initiation revealed >98% clearance of human leukemia cells from the bone marrow, spleen and peripheral blood of mice treated with birinapant doses that achieve drug levels attainable in humans. In vitro apoptosis assays confirmed the greater sensitivity of the Ph-like ALL PDX panel. Moreover, the cIAP1 protein was rapidly degraded in PDXs upon birinapant treatment both in vitro and in vivo, regardless of their relative sensitivity. Microarray analysis of gene expression revealed a significant correlation between baseline TNFα expression and in vivo birinapant sensitivity across 19 PDXs (P = 0.002; R2 = 0.46). While exogenously-added TNFα did not potentiate apoptosis induced by birinapant, a TNFα blocking antibody partially reversed apoptosis in 3/4 Ph-like PDXs. These results show that birinapant exerts profound single-agent in vivo efficacy against Ph-like pediatric ALL PDXs, indicate a role for endogenous TNFα in the birinapant mechanism of action against this high-risk pediatric ALL subtype, and support further evaluation of birinapant in the treatment of Ph-like ALL. Supported by NCI NO1CM42216. Citation Format: Jennifer Richmond, Kathryn Evans, Alissa Robbins, Raushan T. Kurmasheva, Peter J. Houghton, Malcolm A. Smith, Richard B. Lock. In vivo and in vitro efficacy of birinapant in preclinical models of Ph-like pediatric acute lymphoblastic leukemia. [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 1620. doi:10.1158/1538-7445.AM2015-1620

A Single Mouse Trial Platform for Evaluation of Novel Agents in Acute Lymphoblastic Leukemia By the Pediatric Preclinical Testing Consortium

Introduction: Over 70% of infants with acute lymphoblastic leukemia (ALL) present with mixed lineage leukemia (MLL1/KMT2A) gene rearrangements (MLL-r ALL), which is associated with poor prognosis. MLL-r encodes oncogenic fusion proteins that cause aberrant chromatin histone methylation and leukemic transformation of hematopoietic progenitors. A characteristic of MLL-r leukemia is high expression of the FLT3 gene. Menin is critical for leukemic transformation by MLL fusion proteins. VTP-50469, the preclinical precursor of revumenib (SNDX-5613), is a potent and selective small molecule inhibitor of the Menin-MLL interaction that has shown impressive single-agent activity in preclinical models of MLL-r acute leukemia. Revumenib is currently undergoing clinical evaluation, therefore it was of interest to assess the in vivo activity of VTP-50469 in combination with established drugs against MLL-r ALL patient-derived xenografts (PDXs). Methods: VTP-50469 was tested as a single agent against a panel of 7 pediatric MLL-r ALL PDXs in immune-deficient (NSG) mice (120 mg/kg twice daily x 28 days via oral gavage). Combination studies used VTP-50469 formulated in chow (0.1%) available ad libitum for 28 days in combination with either an induction-type regimen of vincristine (0.15 mg/kg once weekly x 4 weeks), dexamethasone (5 mg/kg) and L-asparaginase (1,000 U/kg) both 5 days on 2 days off x 4 weeks, VXL) all administered intraperitoneally, or the FLT3 inhibitor gilteritinib (Xospata, 30 mg/kg x 28 days via oral gavage). Events were defined as >25% huCD45+ in the peripheral blood. Drug efficacy was assessed by event-free survival of treated (T) and control (C) groups by T-C, T/C measurements and stringent objective response criteria (progressive disease, PD; complete response, CR; maintained CR, MCR), and disease infiltration in the spleen and femoral bone marrow (BM) at 28 days post-treatment initiation. Results: Single-agent VTP-50469 elicited MCRs in 6/7 PDXs and significantly (P<0.0001) reduced leukemia infiltration of the spleen (7/7 PDXs) and BM (6/7 PDXs) compared to vehicle control mice. When administered in chow, VTP-50469 elicited a CR in the MLL-7 PDX (T-C 48.2 days, T/C 8.1) and a PD in MLL-8 (T-C 23.0 days, T/C 4.9). VTP-50469 combined with VXL elicited MCRs in both PDXs (MLL-7 T-C 77.0 days, T/C 12.3; MLL-8 T-C 49.1 days, T/C 9.3) and significantly delayed leukemia progression compared with VTP-50469 or VXL alone (P=0.0013). Moreover, VTP-50469 combined with VXL almost completely eradicated leukemia infiltration of spleens and BMs by both PDXs. VTP-50469 combined with gilteritinib did not result in therapeutic enhancement compared to each single agent arm in either PDX. Conclusions: The addition of VTP-50469 to a standard-of-care induction-type regimen (VXL) resulted in therapeutic enhancement against two infant MLL-r ALL PDXs, while VTP-50469 plus gilteritinib was substantially less effective. These results support further clinical evaluation of revumenib in combination with standard-of-care therapy in MLL-r leukemia. Citation Format: Richard B Lock, Kathryn Evans, Sophia Gawan-Taylor, Ben Watts, Timothy Stearns, Eric J Earley, Emily L Jocoy, Carol J Bult, Beverly A Teicher, Gerard M McGeehan, Malcolm A Smith. The menin inhibitor VTP-50469 enhances the in vivo efficacy of established drugs against preclinical models of aggressive infant MLL-r acute lymphoblastic leukemia [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C095.

Chimeric antigen receptor (CAR) T-cell immunotherapies targeting CD19 or CD22 induce remissions in the majority of patients with relapsed/refractory B-cell acute lymphoblastic leukemia (ALL), although relapse due to target antigen loss or downregulation has emerged as a major clinical dilemma. Accordingly, great interest exists in developing CAR T cells directed against alternative leukemia cell surface antigens that may help to overcome immunotherapeutic resistance. The fms-like tyrosine kinase 3 receptor (FLT3) is constitutively activated via FLT3 mutation in acute myeloid leukemia (AML) or wild-type FLT3 overexpression in KMT2A (lysine-specific methyltransferase 2A)-rearranged ALL, which are associated with poor clinical outcomes in children and adults. We developed monovalent FLT3-targeted CAR T cells (FLT3CART) and bispecific CD19xFLT3CART and assessed their anti-leukemia activity in preclinical models of FLT3-mutant AML and KMT2A-rearranged infant ALL. We report robust in vitro FLT3CART-induced cytokine production and cytotoxicity against AML and ALL cell lines with minimal cross-reactivity against normal hematopoietic and non-hematopoietic tissues. We also observed potent in vivo inhibition of leukemia proliferation in xenograft models of both FLT3-mutant AML and KMT2A-rearranged ALL, including a post-tisagenlecleucel ALL-to-AML lineage switch patient-derived xenograft model pairing. We further demonstrate significant in vitro and in vivo activity of bispecific CD19xFLT3CART against KMT2A-rearranged ALL and posit that this additional approach might also diminish potential antigen escape in these high-risk leukemias. Our preclinical data credential FLT3CART as a highly effective immunotherapeutic strategy for both FLT3-mutant AML and KMT2A-rearranged ALL which is poised for further investigation and clinical translation.

Introduction: Vobra duo, a duocarmycin-based humanized ADC (drug-to-antibody ratio is ∼2.7) targeting B7-H3, shows robust in vivo activity against a range of adult cancer models, and a favorable pharmacokinetic and safety profile in cynomolgus monkeys. Initial results from the single agent phase 1 clinical trial of vobra duo (NCT03729596) showed manageable side effects and promising objective response rates in metastatic castration-resistant prostate cancer. B7-H3 is highly expressed in pediatric solid tumors, and it is emerging as a key target for pediatric oncology. Here we report the antitumor activity of vobra duo against preclinical xenograft models of pediatric solid tumors. Methods: Ewing sarcoma (ES), rhabdomyosarcoma (RMS), and neuroblastoma (NB) xenograft models were tested with n=1 or n=2 designs. Osteosarcoma (OS) models, due to their slower growth kinetics and lower rates of tumor regression, were tested with n=10 mice per arm. The study was extended to include malignant rhabdoid tumor (MRT), hepatoblastoma (HB), and Wilms tumor (WT) models using n=5 mice per treatment group. vobra duo and control ADC (SYD988 anti-CD20 ADC with the same linker and payload as vobra duo) were provided by MacroGenics, Inc., and were administered at 6 mg/kg as a single administration IP. Time-to-event was defined as 4-fold increase in tumor xenograft volume from the day of treatment. The Kaplan-Meier method was used to compare event-free survival (EFS) between treated and control groups. Objective response categories were determined as described previously (Ped Blood Cancer 2007;49:928-940), with objective responses including partial, complete, and maintained complete responses (PR, CR, and MCR). Results: Vobra duo induced objective responses in 4 of 11 NB models (3 CR and 1 MCR) and in 3 of 5 OS models (2 MCR). ES models showed objective responses in 2 of 6 models (1 MCR) and 4 of 6 RMS models showed objective responses, all MCR. Control ADC was less active than vobra duo and showed objective responses in 0 of 11 NB, 1 of 5 OS, 1 of 6 ES, and 2 of 4 RMS models. The study was extended to MRT, HB, and WT models. Vobra duo induced objective responses in 2 of 4 MRT with 1 MCR, 2 of 5 HB with 2 MCR, and 3 of 4 WT with 3 MCR. Control ADC only induced objective responses in 1 of 4 MRT and 2 of 4 WT models (all PR), with no objective responses for the 5 HB models. Conclusions: Vobra duo is potently efficacious across a broad panel of pediatric solid tumor xenograft models supporting clinical development of this agent and other agents targeting B7-H3 for children with cancer. Citation Format: Haiying Tang, Edward Favours, Samson Ghilu, Peyton Wong, Vanessa Del Pozo, Abhik Bandyopadhyay, Elena Mironova, Eric J. Earley, Stephen W. Erickson, David Groff, Beverly A. Teicher, Kateryna Krytska, Matthew Tsang, Yael P. Mosse, E. Anders Kolb, Matthew Stephens, Steve Neuhauser, Tim Stearns, Jeff Chuang, Emily L. Jocoy, Jee Young Kwon, Carol Bult, Malcolm A. Smith, John M. Maris, Richard G. Gorlick, Peter J. Houghton, Raushan Kurmasheva. The B7-H3 targeting antibody-drug conjugate (ADC) vobramitamab duocarmazine (vobra duo) is potently effective against a broad panel of pediatric solid tumor xenograft models: A study from the Pediatric Preclinical In Vivo Testing (PIVOT) Consortium [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 7040.

Despite the rarity of each individual cancer type, about 200 different rare cancers constitute in total about 20% of all cancer cases, including pediatric cancers. In Europe, nearly half a million people live with a rare cancer. Like other rare diseases, rare cancers are particular challenging due to their low incidence, particularly for the identification of novel therapies that could improve patient survival. In spite of being the predominant type of pediatric liver malignances, hepatoblastoma (HB), with a world-wide incidence of 1 case per million persons per year, is a rare tumor. Differently from adult hepatocellular carcinoma (HCC) that develops on a cirrhotic or chronically-infected background, liver tumors in children and adolescents occur on apparently normal liver. The high rate (> 60 %) of β-catenin activating mutations places HB as the human tumor most tightly associated with activation of the Wnt/β-catenin pathway. Evidence for (epi)genetic origin of HB is provided by its association with congenital anomalies, Beckwith-Wiedemann syndrome, and familial adenomatous polyposis, a disorder caused by germline mutation of APC, involved in β-catenin degradation. HCC, fibrolamellar carcinoma (FLC), and transitional liver cell tumors (TLCT), which combines histological features of HB and HCC, also arise in children and adolescents, at a lower extent though. Sporadically, very rare forms of liver tumor likely of non-epithelial origin such as rhabdoid tumor or hepatic sarcoma also occur. In order to assist medical decision on the management of liver cancer in childhood and adolescence, we have launched a program aimed at the constitution of liver cancer patient-derived xenografts (PDXs). At present, 8/24 HBs, 2/2 TLCTs, 0/2 FLCs, 1/1 rhabdoid tumor and 0/1 hepatic sarcoma have been successfully grown in immunocompromised mice. HB, TLCT and rhabdoid PDXs maintain the histological features of primary human tumors, and upon treatment with different chemotherapy agents, these models show unique profiles of response, indicating a tumor-specific sensitivity. Given the relatively high number of HB models obtained, HB PDXs could be used as a preclinical cohort for phase II-like studies. This would allow the pre-screen of therapeutic solutions that would require years when not decades to be put in place via standard clinical assays. Moreover, as several HB PDXs harbour activating mutation of β-catenin, they could serve as very powerful tools for the development of efficacious Wnt/β-catenin inhibitors. In addition, for sporadic liver tumors like TLCT and rhabdoid tumor, as the creation of a preclinical cohort is hard to propose, a comprehensive drug screening in vivo could orientate adjuvant therapy in view of a personalized treatment choice, or contribute to accumulate evidence on the usefulness of the tested drugs on such types of liver malignancies. Citation Format: Stefano Cairo, Aurore Gorse, Delphine Nicolle, Erwan Selingue, Frédéric Gauthier, Christophe Chardot, Elie Fadel, Dominique Elias, Daniel Orbach, Catherine Guettier, Arnaud Beurdeley, Vanessa Yvonnet, Olivier Deas, Monique Fabre, Laurence Brugières, Sophie Branchereau, Jean-Gabriel Judde. Liver cancer patient-derived xenografts to improve disease management in childhood and adolescence: perspectives and challenges of personalized medicine. [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 2797. doi:10.1158/1538-7445.AM2013-2797