Xenograft Model Of Acute Lymphoblastic Leukemia Research Articles

Preclinical efficacy of the potent, selective menin-KMT2A inhibitor JNJ-75276617 (bleximenib) in KMT2A- and NPM1-altered leukemias

AbstractThe interaction between menin and histone-lysine N-methyltransferase 2A (KMT2A) is a critical dependency for KMT2A- or nucleophosmin 1 (NPM1)–altered leukemias and an emerging opportunity for therapeutic development. JNJ-75276617 (bleximenib) is a novel, orally bioavailable, potent, and selective protein-protein interaction inhibitor of the binding between menin and KMT2A. In KMT2A-rearranged (KMT2A-r) and NPM1-mutant (NPM1c) acute myeloid leukemia (AML) cells, JNJ-75276617 inhibited the association of the menin-KMT2A complex with chromatin at target gene promoters, resulting in reduced expression of several menin-KMT2A target genes, including MEIS1 and FLT3. JNJ-75276617 displayed potent antiproliferative activity across several AML and acute lymphoblastic leukemia (ALL) cell lines and patient samples harboring KMT2A or NPM1 alterations in vitro. In xenograft models of AML and ALL, JNJ-75276617 reduced leukemic burden and provided a significant dose-dependent survival benefit accompanied by expression changes of menin-KMT2A target genes. JNJ-75276617 demonstrated synergistic effects with gilteritinib in vitro in AML cells harboring KMT2A-r. JNJ-75276617 further exhibited synergistic effects with venetoclax and azacitidine in AML cells bearing KMT2A-r in vitro, and significantly increased survival in mice. Interestingly, JNJ-75276617 showed potent antiproliferative activity in cell lines engineered with recently discovered mutations (MEN1M327I or MEN1T349M) that developed in patients refractory to the menin-KMT2A inhibitor revumenib. A cocrystal structure of menin in complex with JNJ-75276617 indicates a unique binding mode distinct from other menin-KMT2A inhibitors, including revumenib. JNJ-75276617 is being clinically investigated for acute leukemias harboring KMT2A or NPM1 alterations, as a monotherapy for relapsed/refractory acute leukemia (NCT04811560), or in combination with AML-directed therapies (NCT05453903).

Combination p53 activation and BCL-xL/BCL-2 inhibition as a therapeutic strategy in high-risk and relapsed acute lymphoblastic leukemia

Due to the rarity of TP53 mutations in acute lymphoblastic leukemia (ALL), p53 re-activation by antagonism of the p53-MDM2 interaction represents a potential therapeutic strategy for the majority of ALL. Here, we demonstrate the potent antileukemic activity of the MDM2 antagonist idasanutlin in high-risk and relapsed ex vivo coculture models of TP53 wildtype ALL (n = 40). Insufficient clinical responses to monotherapy MDM2 inhibitors in other cancers prompted us to explore optimal drugs for combination therapy. Utilizing high-throughput combination screening of 1971 FDA-approved and clinically advanced compounds, we identified BCL-xL/BCL-2 inhibitor navitoclax as the most promising idasanutlin combination partner. The idasanutlin-navitoclax combination was synergistically lethal to prognostically-poor, primary-derived and primary patient blasts in ex vivo coculture, and reduced leukemia burden in two very high-risk ALL xenograft models at drug concentrations safely attained in patients; in fact, the navitoclax plasma concentrations were equivalent to those attained in contemporary “low-dose” navitoclax clinical trials. We demonstrate a preferential engagement of cell death over G1 cell cycle arrest, mechanistically implicating MCL-1-binding pro-apoptotic sensitizer NOXA. The proposed combination of two clinical-stage compounds independently under clinical evaluation for ALL is of high clinical relevance and warrants consideration for the treatment of patients with high-risk and relapsed ALL.

Restoration of LAT Signaling in CD33-Directed CAR T Cells Improves Potency and Persistence Against Acute Myeloid Leukemia

Despite the development of novel therapies, the majority of patients with acute myeloid leukemia (AML) will not achieve long-term remissions. Patients with relapsed/refractory (r/r) AML have particularly poor outcomes, as conventional chemotherapy regimens are often unable to elicit long-term remissions. Allogeneic hematopoietic stem cell transplant (HSCT) is potentially curative in eligible patients, however, bridging therapies which can induce deep remissions prior to HSCT are needed. Second generation chimeric antigen receptor (CAR) T cells targeting lineage-restricted antigens, such as CD19, have induced remissions in up to 80 - 90% of pediatric patients with r/r B-lineage acute lymphoblastic leukemia (ALL), however, post-CAR relapses are common and only ~50% of treated patients achieve durable remissions. Similar strategies targeting myeloid-restricted antigens with 2 nd generation CAR T cells in AML have not replicated the success seen in ALL. As clinical experience has highlighted the impact of CAR T cell potency, persistence, and antigen-sensitivity to long-term efficacy in ALL, strategies to boost these properties in myeloid-directed CAR T cells are necessary to extend the clinical benefit of this therapy to patients with AML. Utilizing a global phosphoproteomics screen, we recently identified that inefficient phosphorylation of the Linker for activation of T cells (LAT) and the subsequent decrease in the downstream signaling pathways results in suboptimal CAR T cell activation. To overcome inefficient LAT activation, we developed a novel bicistronic CAR platform, consisting of a 2 nd generation CAR along with an Adjunctive LAT-Activating CAR (ALA-CART) which improved sensitivity to low-antigen leukemia cells, increased in vivo potency, and enhanced persistence in xenograft models of ALL. We hypothesized that the restoration of LAT signaling via the ALA-CART platform would also improve potency and persistence in pre-clinical models of AML. We found that while CD33 ALA-CART cells responded to CD33+ AML cells in vitro, they were less cytotoxic and produced lower levels of cytokines than CD33-28z or CD33-BBz 2 ndgeneration CAR T cells. However, CD33 ALA-CART cells demonstrated increased potency in xenograft models, eradicating AML cells at much lower doses than CD33-28z and CD33-BBz CAR T cells (Fig. 1A, p<0.0001). This in vivo potency correlated with an enrichment of T stem cell memory cells in the ALA-CART product, resulting in increased persistence of CD33 ALA-CART cells after leukemia clearance (Fig. 1B, p=0.0139). The ALA-CART platform was associated with increased mitochondrial mass and function, suggesting that the increased signaling through LAT results in an enhanced capacity for oxidative phosphorylation, potentially contributing to the increased persistence of CD33 ALA-CART cells in vivo. Thus, the ALA-CART platform, designed to restore LAT signaling in CAR T cells, demonstrates that CAR molecules can be rationally designed to enhance both in vivo potency and persistence, and represents a promising strategy for the development for more effective AML-directed CAR T cells.

Establishment of a Patient-Derived T-Cell Acute Lymphoblastic Leukemia Xenograft Model in Novel Immunodeficient NCG Mice

The leukemia cells from patients with T-cell acute lymphoblastic leukemia (T-ALL) were inoculated into NCG mice to establish a stable human T-ALL leukemia animal model. Leukemia cells from bone marrow of newly diagnosed T-ALL patients were isolated, and the leukemia cells were inoculated into NCG mice via tail vein. The proportion of hCD45 positive cells in peripheral blood of the mice was detected regularly by flow cytometry, and the infiltration of leukemia cells in bone marrow, liver, spleen and other organs of the mice was detected by pathology and immunohistochemistry. After the first generation mice model was successfully established, the spleen cells from the first generation mice were inoculated into the second generation mice, and after the second generation mice model was successfully established, the spleen cells from the second generation mice were further inoculated into the third generation mice, and the growth of leukemia cells in peripheral blood of the mice in each group was monitored by regular flow cytometry to evaluate the stability of this T-ALL leukemia animal model. On the 10th day after inoculation, hCD45+ leukemia cells could be successfully detected in the peripheral blood of the first generation mice, and the proportion of these cells was gradually increased. On average, the mice appeared listless 6 or 7 weeks after inoculation, and a large number of T lymphocyte leukemia cells were found in the peripheral blood and bone marrow smear of the mice. The spleen of the mice was obviously enlarged, and immunohistochemical examination showed that hCD3+ leukemia cells infiltrated into bone marrow, liver and spleen extensively. The second and third generation mice could stably develop leukemia, and the average survival time was 4-5 weeks. Inoculating leukemia cells from bone marrow of patients with T-ALL into NCG mice via tail vein can successfully construct a patient-derived tumor xenografts (PDTX) model.

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

Introduction: Children diagnosed with acute lymphoblastic leukemia (ALL) experience close to a 90% likelihood of cure, however the outcome for high-risk subtypes remains poor. Evasion of apoptosis is a hallmark of cancer, with dysregulation of the BCL-2 family of proteins often reported (Delbridge et al, Nat Rev Cancer 16:99-109, 2016). The BCL-2 family consists of the anti-apoptotic MCL1, BCL-2 and BCLXL as well as pro-apoptotic proteins. AMG 176, a first-in-class MCL1 inhibitor, previously demonstrated potent apoptosis-inducing activity in the nanomolar range against cell lines and patient explants of human hematological malignancies (Caenepeel et al, Cancer Discov 8:1582-97, 2018). Furthermore, discontinuous oral administration of AMG 176 inhibited the growth of human acute myeloid leukemia and multiple myeloma tumor xenografts in vivo at well-tolerated doses. AMG 176 has entered clinical evaluation for patients with hematological malignancies. Therefore, it was of interest for the Pediatric Preclinical Testing Consortium (PPTC) to evaluate the in vivo efficacy of AMG 176 against its patient-derived xenograft (PDX) models of pediatric ALL. Methods:MCL1, BCL-2 and BCLXL mRNA expression was quantified by RNA-seq (http://pedcbioportal.kidsfirstdrc.org). For in vitro cytotoxicity assays, cells were treated with AMG 176 (10 μM - 1 pM) or DMSO control for 24 h and cell viability was determined by AlamarBlue assay and expressed as IC50values. AMG 176 was evaluated in vivo against a panel of 38 ALL PDXs in a single mouse trial format. Each PDX was inoculated via tail vein injection into one NSG mouse (1-5 x 106 cells per mouse). Engraftment and drug responses were determined by enumerating the proportion of human versus mouse CD45+ (%huCD45+) cells in the peripheral blood (PB) at weekly intervals. Treatment with AMG 176 (provided by Amgen, Inc.) began once the %huCD45+ was ≥1% in each mouse and consisted of 60 mg/kg via oral gavage 2 days on, 5 days off for 2 weeks. The experimental endpoint was pre-defined as the %huCD45+ reaching 25% in the PB. Drug efficacy was assessed by event-free survival (EFS) and stringent objective response measures (Houghton et al, Pediatr Blood Cancer 49:928-40, 2007). For the pharmacokinetic (PK) study naïve NSG mice were treated with either vehicle or AMG 176 (60 mg/kg) and groups of 3 mice harvested up to 24 h post-treatment. Plasma samples were collected for analysis of AMG 176 using acetonitrile protein precipitation and LC-MS/MS with gradient elution on a Phenomenex Kinetex XB-C18 (3.0 x 50 mm, 2.6 µm) analytical column. Results:MCL1 mRNA expression ranged from 21-282 FPKM across a panel of 90 ALL PDXs representing 6 broad ALL subtypes (B-cell precursor, BCP-ALL; Ph+-ALL; Ph-like ALL; mixed-lineage leukemia-rearranged, MLLr-ALL; T-cell ALL, T-ALL; and early T-cell precursor ALL, ETP-ALL). Thirty eight of the 90 ALL PDXs were selected for in vivo evaluation of AMG 176 activity based on their profiles of MCL1, BCL-2 and BCLXL mRNA expression. AMG 176 was well tolerated in the efficacy study, with only 3 mice experiencing >10% weight loss. Mouse EFS values ranged from 3.5 to 47.8 days from treatment initiation, however no PDX achieved an objective response (remission) with all but one PDX exhibiting progressive disease. The Ph-like ALL PDX, ALL-102 (MCL1 expression 71.8 FPKM) scored a stable disease and had the highest EFS value (47.8 days). In an effort to explain the poor in vivo activity of AMG 176 several follow-up studies were performed. First, the in vitro IC50 values elicited by the AMG-176 stock against 5 leukemia cell lines (MOLM-13, 1.6 µM; NB-4, 2.0 µM; Kasumi-1, 1.6 µM; THP-1, 1.2 µM; and U-937,1.3 µM) were comparable to published data. Second, C18 LC-MS analysis revealed major and minor peaks consistent with theoretical peaks with negligible degradation. Third, PK study results demonstrated that the AMG 176 plasma concentration peaked at 8.5 µM, one hour after administration, with an AUC of 87.7 µM*hr and t1/2 of 4.1 h. This AUC was markedly lower than that in athymic nude mice treated at the same dose (1,120 µM*hr). Conclusions: AMG 176 exhibited modest in vivo activity against a large range of pediatric ALL PDXs, with the best response defined as stable disease in a single PDX. PK data suggest that AMG 176 systemic exposures in NSG mice may not be sufficient to elicit meaningful and prolonged regressions in pediatric ALL PDXs. (Supported by NCI Grants CA199000 and CA199222)

Epigenetic activation of the FLT3 gene by ZNF384 fusion confers a therapeutic susceptibility in acute lymphoblastic leukemia

FLT3 is an attractive therapeutic target in acute lymphoblastic leukemia (ALL) but the mechanism for its activation in this cancer is incompletely understood. Profiling global gene expression in large ALL cohorts, we identify over-expression of FLT3 in ZNF384-rearranged ALL, consistently across cases harboring different fusion partners with ZNF384. Mechanistically, we discover an intergenic enhancer element at the FLT3 locus that is exclusively activated in ZNF384-rearranged ALL, with the enhancer-promoter looping directly mediated by the fusion protein. There is also a global enrichment of active enhancers within ZNF384 binding sites across the genome in ZNF384-rearranged ALL cells. Downregulation of ZNF384 blunts FLT3 activation and decreases ALL cell sensitivity to FLT3 inhibitor gilteritinib in vitro. In patient-derived xenograft models of ZNF384-rearranged ALL, gilteritinib exhibits significant anti-leukemia efficacy as a monotherapy in vivo. Collectively, our results provide insights into FLT3 regulation in ALL and point to potential genomics-guided targeted therapy for this patient population.

The first Japanese biobank of patient-derived pediatric acute lymphoblastic leukemia xenograft models.

A lack of practical resources in Japan has limited preclinical discovery and testing of therapies for pediatric relapsed and refractory acute lymphoblastic leukemia (ALL), which has poor outcomes. Here, we established 57 patient-derived xenografts (PDXs) in NOD.Cg-Prkdcscid ll2rgtm1Sug /ShiJic (NOG) mice and created a biobank by preserving PDX cells including three extramedullary relapsed ALL PDXs. We demonstrated that our PDX mice and PDX cells mimicked the biological features of relapsed ALL and that PDX models reproduced treatment-mediated clonal selection. Our PDX biobank is a useful scientific resource for capturing drug sensitivity features of pediatric patients with ALL, providing an essential tool for the development of targeted therapies.

Potent preclinical activity of FLT3-directed chimeric antigen receptor T-cell immunotherapy against FLT3- mutant acute myeloid leukemia and KMT2A-rearranged acute lymphoblastic leukemia.

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 KMT2Arearranged 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.

Abstract 3934: KT-253, a highly potent and selective heterobifunctional MDM2 degrader for the treatment of wildtype p53 tumors with superior potency and differentiated biological activity compared to small molecule inhibitors (SMI)

Abstract Background: The murine double minute 2 (MDM2) oncoprotein is a key E3 ubiquitin ligase that degrades the tumor-suppressor p53. Reversible SMIs of the MDM2/p53 interaction have been developed to stabilize p53 and to induce apoptosis in wildtype p53 tumors. However, MDM2 SMIs induce a p53/MDM2 feedback loop, resulting in upregulation of MDM2 protein levels and p53 pathway inhibition, thus drastically limiting their biological activity and clinical application. MDM2 targeted protein degradation suppresses p53-dependent MDM2 protein feedback upregulation and is therefore expected to lead to a superior response compared to SMIs. Objectives: We discovered KT-253, a novel, highly potent heterobifunctional MDM2 degrader. We characterized the activity and molecular mechanism of KT-253 in wildtype p53 cell lines and xenograft models and demonstrated that KT-253 has superior activity compared to SMIs in these models. Methods: We characterized KT-253 in MDM2-dependent cell lines in vitro, as well as in in vivo mouse AML and ALL xenograft models. Methods included in vitro cell proliferation and apoptosis assays, gene expression profiling, and in vivo pharmacological studies. Results: KT-253 selectively depletes MDM2 protein levels with sub-nanomolar cellular potency. We demonstrated in the Acute Lymphoblastic Leukemia (ALL) cell line RS4;11 that KT-253 can overcome the p53-dependent upregulation of MDM2 protein levels as seen for reversible SMIs. In addition, even brief exposures to KT-253 in these cells could more potently stabilize p53 compared to SMIs, leading to apoptosis mediated through p53 target genes. The superior MDM2/p53 pathway inhibition and induction of apoptosis by KT-253 translates into a >200-fold stronger cell growth inhibition, compared to SMIs, across a panel of solid and hematological tumor cell lines. Using mouse xenograft models, we established an intermittent dosing schedule that drives anti-tumor efficacy. Importantly, a single dose of KT-253 at 1 mg/kg resulted in sustained tumor regression in the RS4;11 mouse xenograft model. In this model, KT-253 exposures correlated with induction of apoptotic p53 target genes and tumor growth inhibition. In addition, weekly administration of KT-253 significantly prolonged the survival in a disseminated Acute Myeloid Leukemia (AML) model when compared to vehicle treated animals. Conclusions: In summary, KT-253, our development candidate, has significantly improved potency relative to reversible SMIs leading to potent in vitro and in vivo efficacy that is superior to all clinically active agents. In addition, an intermittent dosing schedule of the highly potent KT-253 degrader can induce rapid apoptosis in MDM2-dependent cancer cells potentially leading to improved efficacy and safety profile. Citation Format: Yogesh Chutake, Michele Mayo, Dapeng Chen, Bradley Enerson, Patricia Cho, Jessica Filiatrault, Crystal Brown, Michael Placke, Madison Adams, Rahul Karnik, James Shaw, Yatao Shi, Dirk Walther, Alice McDonald, Frank Qi, Phillip Liu, Joseph D. Growney, Kirti Sharma, Duncan Walker, Stefanie Schalm, Juliet Williams, Matthew Weiss. KT-253, a highly potent and selective heterobifunctional MDM2 degrader for the treatment of wildtype p53 tumors with superior potency and differentiated biological activity compared to small molecule inhibitors (SMI) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3934.

Abstract 5571: Disruption of cell-intrinsic checkpoint regulator CTLA-4 in CD19 directed CAR T cells provides clinical efficacy in CLL patients

Abstract The field of cancer immunotherapy has led to successful treatments such as monoclonal antibodies (MAb), inhibitory receptor (IR) blockade, and adoptive cell transfer (ACT). ACT modified to express chimeric antigen receptors (CARs) can redirect T cells to tumor antigens and has shown remarkable clinical efficacy in patients with relapse and refractory leukemia and lymphoma. 90% of pediatric patients with acute lymphoblastic leukemia (ALL) respond to CD19 CAR T cell therapy (CAR T19), although only 26-35% of patients with chronic lymphocytic leukemia (CLL) show complete responses (CR). It is unclear why responses are less frequent in CLL compared to ALL. The heavy pre-treatment received by CLL patients likely contributes to lower response rates with CAR T cell therapy, and disease progression is worsened due to profound T cell defects characterized by elevated expression of IRs such as PD-1 and CTLA-4. Fraietta et. al. assessed CLL patient apheresis samples and CART19 products to show that CR was associated with elevated levels of CD27+PD1-CD8+ T memory cells whereas the non-responders (NR) showed an exhausted phenotype with high levels of multiple IRs. We studied 14 patients with advanced, heavily pretreated CLL who received at least one dose of CART19. Patients with CRs exhibited high in vivo expansion and persistence of infused CAR T cells, as opposed to NR’s. Importantly, at peak levels of in vivo CAR expansion, NR’s had elevated levels of CTLA-4 expression which correlated with poor CLL patient responses to CART19 therapy. In summary, these data suggest that eliminating CTLA-4 mediated T cell inhibition can be clinically beneficial. Thus, we tested the hypothesis that disruption of CTLA4 would improve CAR T cell efficacy in CLL based on numerous observations. Our data demonstrate that knockout (KO) of CTLA-4 in T cells from healthy donors using CRISPR Cas9 technology leads to maintenance of surface CAR expression and higher tumor clearance in a chronic re-stimulation model using CART19 cells against NALM6 tumor cells. In xenograft models of ALL, KO of CTLA-4 increases the anti-tumor efficacy of CART19 cells. We then performed CTLA4 disruption in T cells banked from CLL patients that did not respond to CAR T cell therapy to determine whether dysfunctional CAR T infusion products can be invigorated by CTLA-4 KO. In both chronic re-stimulation model and xenograft models of NALM6, CTLA-4 KO CD19 CAR T cells from CLL patients maintained surface CAR expression, exhibited enhanced tumor clearance, and higher survival rates relative to subject-matched unedited CAR T cells. Thus, suggesting that CTLA4 disrupted CAR T cell products may enhance the success rate of CAR T cell therapy for CLL patients. This technology can be feasibly expanded to other tumor indications to increase the overall efficacy of CAR T cells. These IND-enabling studies will support the translation of this therapy to the clinic. Citation Format: Sangya Agarwal, Angela Aznar Gomez, Tong Da, Shunichiro Kuramitsu, Weimin Kong, Pranali Ravikumar, Mercy Gohil, Megan M. Davis, Joseph A. Fraietta, Gabriela Plesa, David L. Porter, Regina M. Young, Carl H. June. Disruption of cell-intrinsic checkpoint regulator CTLA-4 in CD19 directed CAR T cells provides clinical efficacy in CLL patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5571.

Abstract LB171: The differentiated CD47 monoclonal antibody AO-176 exhibits significant in vivo activity against xenograft models of pediatric acute lymphoblastic leukemia (ALL)

Abstract 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.

Abstract 3039: Pediatric preclinical testing consortium evaluation of the dual SYK/FLT3 inhibitor TAK-659 in xenograft models of pediatric acute lymphoblastic leukemia

Abstract 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.

Irinotecan Induces Disease Remission in Xenograft Mouse Models of Pediatric MLL-Rearranged Acute Lymphoblastic Leukemia.

Acute lymphoblastic leukemia (ALL) in infants (<1 year of age) remains one of the most aggressive types of childhood hematologic malignancy. The majority (~80%) of infant ALL cases are characterized by chromosomal translocations involving the MLL (or KMT2A) gene, which confer highly dismal prognoses on current combination chemotherapeutic regimens. Hence, more adequate therapeutic strategies are urgently needed. To expedite clinical transition of potentially effective therapeutics, we here applied a drug repurposing approach by performing in vitro drug screens of (mostly) clinically approved drugs on a variety of human ALL cell line models. Out of 3685 compounds tested, the alkaloid drug Camptothecin (CPT) and its derivatives 10-Hydroxycamtothecin (10-HCPT) and 7-Ethyl-10-hydroxycamtothecin (SN-38: the active metabolite of the drug Irinotecan) appeared most effective at very low nanomolar concentrations in all ALL cell lines, including models of MLL-rearranged ALL (n = 3). Although the observed in vitro anti-leukemic effects of Camptothecin and its derivatives certainly were not specific to MLL-rearranged ALL, we decided to further focus on this highly aggressive type of leukemia. Given that Irinotecan (the pro-drug of SN-38) has been increasingly used for the treatment of various pediatric solid tumors, we specifically chose this agent for further pre-clinical evaluation in pediatric MLL-rearranged ALL. Interestingly, shortly after engraftment, Irinotecan completely blocked leukemia expansion in mouse xenografts of a pediatric MLL-rearranged ALL cell line, as well as in two patient-derived xenograft (PDX) models of MLL-rearranged infant ALL. Also, from a more clinically relevant perspective, Irinotecan monotherapy was able to induce sustainable disease remissions in MLL-rearranged ALL xenotransplanted mice burdened with advanced leukemia. Taken together, our data demonstrate that Irinotecan exerts highly potent anti-leukemia effects against pediatric MLL-rearranged ALL, and likely against other, more favorable subtypes of childhood ALL as well.

Degradation of Janus kinases in CRLF2-rearranged acute lymphoblastic leukemia

AbstractCRLF2-rearranged (CRLF2r) acute lymphoblastic leukemia (ALL) accounts for more than half of Philadelphia chromosome-like (Ph-like) ALL and is associated with a poor outcome in children and adults. Overexpression of CRLF2 results in activation of Janus kinase (JAK)-STAT and parallel signaling pathways in experimental models, but existing small molecule inhibitors of JAKs show variable and limited efficacy. Here, we evaluated the efficacy of proteolysis-targeting chimeras (PROTACs) directed against JAKs. Solving the structure of type I JAK inhibitors ruxolitinib and baricitinib bound to the JAK2 tyrosine kinase domain enabled the rational design and optimization of a series of cereblon (CRBN)-directed JAK PROTACs utilizing derivatives of JAK inhibitors, linkers, and CRBN-specific molecular glues. The resulting JAK PROTACs were evaluated for target degradation, and activity was tested in a panel of leukemia/lymphoma cell lines and xenograft models of kinase-driven ALL. Multiple PROTACs were developed that degraded JAKs and potently killed CRLF2r cell lines, the most active of which also degraded the known CRBN neosubstrate GSPT1 and suppressed proliferation of CRLF2r ALL in vivo, e.g. compound 7 (SJ988497). Although dual JAK/GSPT1-degrading PROTACs were the most potent, the development and evaluation of multiple PROTACs in an extended panel of xenografts identified a potent JAK2-degrading, GSPT1-sparing PROTAC that demonstrated efficacy in the majority of kinase-driven xenografts that were otherwise unresponsive to type I JAK inhibitors, e.g. compound 8 (SJ1008030). Together, these data show the potential of JAK-directed protein degradation as a therapeutic approach in JAK-STAT–driven ALL and highlight the interplay of JAK and GSPT1 degradation activity in this context.

Doxorubicin/Nucleophosmin Binding Protein-Conjugated Nanoparticle Enhances Anti-leukemia Activity in Acute Lymphoblastic Leukemia Cells in vitro and in vivo.

Acute lymphoblastic leukemia (ALL) is an aggressive malignancy. Adults with ALL have more than 50% relapse rates. We have previously validated that overexpression of nucleophosmin (NPM) is involved in the multidrug resistance (MDR) development during ALL; and a synthetically engineered recombinant NPM binding protein (NPMBP) has been developed in our group; NPMBP and doxorubicin (DOX) can be conjugated in a nanoparticle-based drug delivery system named DOX-PMs-NPMBP to counteract MDR during ALL. Here, we evaluated the antileukemia potential of DOX-PMs-NPMBP in resistant ALL cells. This study demonstrates that DOX-PMs-NPMBP significantly enhances chemosensitivity to DOX in ALL cells. Despite at variable concentrations, both resistant and primary ALL cells from relapsed patients were sensitive to DOX-PMs-NPMBP. In detail, the half maximal inhibitory concentration (IC50) values of DOX-PMs-NPMBP were between 1.6- and 7.0-fold lower than those of DOX in cell lines and primary ALL cells, respectively; and apoptotic cells ratio was over 2-fold higher in DOX-PMs-NPMBP than DOX. Mechanistically, p53-driven apoptosis induction and cell cycle arrest played essential role in DOX-PMs-NPMBP-induced anti-leukemia effects. Moreover, DOX-PMs-NPMBP significantly inhibited tumor growth and prolonged mouse survival of ALL xenograft models; and no systemic toxicity occurrence was observed after treatment during follow-up. In conclusion, these data indicate that DOX-PMs-NPMBP may significantly exert growth inhibition and apoptosis induction, and markedly improve DOX antileukemia activity in resistant ALL cells. This novel drug delivery system may be valuable to develop as a new therapeutic strategy against multidrug resistant ALL.

Targeting TSLP-Induced Tyrosine Kinase Signaling Pathways in CRLF2-Rearranged Ph-like ALL.

Philadelphia (Ph)-like acute lymphoblastic leukemia (ALL) is characterized by aberrant activation of signaling pathways and high risk of relapse. Approximately 50% of Ph-like ALL cases overexpress cytokine receptor-like factor 2 (CRLF2) associated with gene rearrangement. Activated by its ligand thymic stromal lymphopoietin (TSLP), CRLF2 signaling is critical for the development, proliferation, and survival of normal lymphocytes. To examine activation of tyrosine kinases regulated by TSLP/CRLF2, phosphotyrosine (P-Tyr) profiling coupled with stable isotope labeling of amino acids in cell culture (SILAC) was conducted using two CRLF2-rearranged (CRLF2r) Ph-like ALL cell lines stimulated with TSLP. As a result, increased P-Tyr was detected in previously reported TSLP-activated tyrosine kinases and substrates, including JAK1, JAK2, STAT5, and ERK1/2. Interestingly, TSLP also increased P-Tyr of insulin growth factor 1 receptor (IGF1R) and fibroblast growth factor receptor 1 (FGFR1), both of which can be targeted with small-molecule inhibitors. Fixed-ratio combination cytotoxicity assays using the tyrosine kinase inhibitors BMS-754807 and ponatinib that target IGF1R and FGFR1, respectively, revealed strong synergy against both cell line and patient-derived xenograft (PDX) models of CRLF2r Ph-like ALL. Further analyses also indicated off-target effects of ponatinib in the synergy, and novel association of the Ras-associated protein-1 (Rap1) signaling pathway with TSLP signaling in CRLF2r Ph-like ALL. When tested in vivo, the BMS-754807/ponatinib combination exerted minimal efficacy against 2 Ph-like ALL PDXs, associated with low achievable plasma drug concentrations. Although this study identified potential new targets in CRLF2r Ph-like ALL, it also highlights that in vivo validation of synergistic drug interactions is essential. IMPLICATION: Quantitative phosphotyrosine profiling identified potential therapeutic targets for high-risk CRLF2-rearranged Ph-like ALL.

Adriamycin/Nucleophosmin Binding Protein-Conjugated Nanoparticle (ADR-PMs-NPMBP) Enhances Anti-Leukemia Activities of Adriamycin in Acute Lymphoblastic Leukemia Cells

Acute lymphoblastic leukemia (ALL) is an aggressive malignancy. Adults with ALL have more than 50% relapse rates and 20-40% overall survival. We previously validated that overexpression of NPM may be involved in the multidrug resistance development and might be important indicator for prognosis evaluation in ALL. In this study, we engineered and synthesized recombinant NPM binding protein (NPMBP). NPMBP and adriamycin (ADR) were bound to polymeric nanoparticle-based drug delivery system and named ADR-PMs-NPMBP (China Patent Application 202010302395.6, 202010739575.0). In this study, we aimed to evaluate thein vitroandin vivoanti-leukemia activity of ADR-PMs-NPMBP in resistant ALL cells. The underlying mechanisms were further addressed. In vitrostudy data demonstrated that ADR-PMs-NPMBP significantly enhanced chemosensitivity of ALL cells to ADR. The IC50 values of ADR-PMs-NPMBP in 4 ALL cell lines and primary ALL cells from 6 patients were between 1.6- and 7.0-fold lower than those of ADR. Of note, all the resistant ALL cell lines and the primary ALL cells from the relapsed patients, although variable in degree, were sensitive to ADR-PMs-NPMBP. The ratio of apoptosis cells in resistant ALL cells was more than 2-fold as compared ADR-PMs-NPMBP with single ADR regimen. RNA-seq analysis showed that there were 5599 and 233 differentially expressed genes in ADR-PMs-NPMBP versus control and ADR-PMs-NPMBP versus ADR comparison groups, respectively. Kyoto Encyclopedia of Genes and Genomes enrichment analysis indicated that 21 pathways play significant roles in ADR-PMs-NPMBP-mediated anti-leukemia activities in ALL. Of note, p53 protein with gene symbol TP53 was interestingly identified to overlap in the 5 pathways as following: Epstein-Barr virus infection, cell cycle, apoptosis, human T-cell leukemia virus 1 infection and p53 signaling pathway. Further validation experiments showed that ADR-PMs-NPMBP-induced effects on cell viability were due to the multiple roles of the activated p53 in apoptosis induction and cell cycle arrest. In vivostudy showed that ADR-PMs-NPMBP regimen was well tolerated by both resistant ALL xenograft BALB/C-nude model and ALL cell transplanted NOD-Prkdcem26Il2rgem26Nju (NCG) model. No systemic toxicity occurrence was observed after the recipients were intraperitoneally administered with ADR-PMs-NPMBP at 25 mg/kg/day for 3 days. The body weights of the animals increased with the time in both tumor model systems. Of note, whole-body bioluminescence imaging showed that less tumor cell burden was observed in ADR-PMs-NPMBP-treated recipients as compared to the animals administered with ADR. The tumor inhibition rate achieved as high as 79.2% in ADR-PMs-NPMBP group when finished the following-up. Moreover, studies on NCG model showed that ADR-PMs-NPMBP conditioning sensitized the antileukemia response of the resistant ALL xenograft model to ADR. Wright-Giemsa staining results demonstrated that more mature myeloid cells and lymphocytes were presented in the bone marrow samples from ADR-PMs-NPMBP-conditioned mice. In contrast, the increased immature blast cells were observed in the bone marrow from vehicle control mice and ADR-treated mice. Notably, the regimen of ADR-PMs-NPMBP diminished or blocked simultaneous metastases to multiple organs including liver and spleen, and significantly prolonged mouse survival. In conclusion, our study provides the first evidence that ADR-PMs-NPMBP nanoparticle may exert apoptotic inductionviathe activation of p53 pathway and markedly improve the antileukemia effect of ADR in resistant ALL cellsin vitroandin vivo. Our data indicate that the novel drug delivery system may be valuable to develop as a new strategy for the treatment of ALL, particularly for the resistant ALL. Disclosures No relevant conflicts of interest to declare.

Endogenous TCR promotes in vivo persistence of CD19-CAR-T cells compared to a CRISPR/Cas9-mediated TCR knockout CAR

AbstractAnti-CD19 chimeric antigen receptor (CAR) T cells showed significant antileukemic activity in B-precursor acute lymphoblastic leukemia (ALL). Allogeneic, HLA-mismatched off-the-shelf third-party donors may offer ideal fitness of the effector cells, but carry the risk of graft-versus-host disease. Knockout (KO) of the endogenous T-cell receptor (TCR) in CD19-CAR-T cells may be a promising solution. Here, we induced a CRISPR/Cas9-mediated KO of the TCRβ chain in combination with a second-generation retroviral CAR transduction including a 4-1BB costimulatory domain in primary T cells. This tandem engineering led to a highly functional population of TCR-KO-CAR-T cells with strong activation (CD25, interferon γ), proliferation, and specific killing upon CD19 target recognition. TCR-KO-CAR-T cells had a balanced phenotype of central memory and effector memory T cells. KO of the endogenous TCR in T cells strongly ablated alloreactivity in comparison with TCR-expressing T cells. In a patient-derived xenograft model of childhood ALL, TCR-KO-CAR-T cells clearly controlled CD19+ leukemia burden and improved survival in vivo. However, coexpression of endogenous TCR plus CAR led to superior persistence of T cells and significantly prolonged leukemia control in vivo, confirmed by a second in vivo model using the leukemia cell line NALM6. These results point toward an essential role of the endogenous TCR for longevity of the response at the price of alloreactivity. In conclusion, anti-CD19 CAR T cells with a CRISPR/Cas9-mediated TCR-KO are promising candidates for nonmatched third-party adoptive T-cell transfer with high antileukemic functionality in the absence of alloreactivity, but long-term persistence in vivo is better in the presence of the endogenous TCR.

Drug Elimination Alteration in Acute Lymphoblastic Leukemia Mediated by Renal Transporters and Glomerular Filtration.

Drug elimination alteration has been well reported in acute lymphoblastic leukemia (ALL). Considering that transporters and glomerular filtration influence, to different extents, the drug disposition, and possible side effects, we evaluated the effects of ALL on major renal transporters and glomerular filtration mediated pharmacokinetic changes, as well as expression of renal drug transporters. ALL xenograft models were established and intravenously injected with substrates of renal transporters and glomerular filtration separately in NOD/SCID mice. The plasma concentrations of substrates, after single doses, were determined using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). With the development of ALL, protein expression of MDR1, OAT3 and OCT2 were increased by 2.62-fold, 1.70-fold, and 1.45-fold, respectively, whereas expression of MRP2 and MRP4 were significantly decreased by 30.98% and 45.28% in the kidney of ALL groups compared with control groups. Clearance of MDR1-mediated digoxin, OAT3-mediated furosemide, and OCT2-mediated metformin increased by 3.04-fold, 1.47-fold, and 1.26-fold, respectively. However, clearance of MRPs-mediated methotrexate was reduced by 39.5%. These results are consistent with mRNA expression. Clearance of vancomycin and amikacin, as markers of glomerular filtration rate, had a 2.14 and 1.64-fold increase in ALL mice, respectively. The specific alteration of renal transporters and glomerular filtration in kidneys provide a rational explanation for changes in pharmacokinetics for ALL.

Abstract C004: The CXCR4 inhibitor X4-136 enhances the in vivo efficacy of established drugs against preclinical models of aggressive pediatric acute lymphoblastic leukemia

Abstract 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&amp;lt;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&amp;lt;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&amp;lt;0.05) and ALL-31 (P&amp;lt;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&amp;lt;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