Preclinical Patient-derived Xenograft Models Research Articles

Glutamine antagonist DRP-104 suppresses tumor growth and enhances response to checkpoint blockade in KEAP1 mutant lung cancer.

Loss-of-function mutations in KEAP1 frequently occur in lung cancer and are associated with poor prognosis and resistance to standard of care treatment, highlighting the need for the development of targeted therapies. We previously showed that KEAP1 mutant tumors consume glutamine to support the metabolic rewiring associated with NRF2-dependent antioxidant production. Here, using preclinical patient-derived xenograft models and antigenic orthotopic lung cancer models, we show that the glutamine antagonist prodrug DRP-104 impairs the growth of KEAP1 mutant tumors. We find that DRP-104 suppresses KEAP1 mutant tumors by inhibiting glutamine-dependent nucleotide synthesis and promoting antitumor T cell responses. Using multimodal single-cell sequencing and ex vivo functional assays, we demonstrate that DRP-104 reverses T cell exhaustion, decreases Tregs, and enhances the function of CD4 and CD8 T cells, culminating in an improved response to anti-PD1 therapy. Our preclinical findings provide compelling evidence that DRP-104, currently in clinical trials, offers a promising therapeutic approach for treating patients with KEAP1 mutant lung cancer.

Abstract 2098: A novel GCN2 kinase activator demonstrates therapeutic efficacy in preclinical PDX models of human cholangiocarcinoma

Abstract Introduction Cholangiocarcinoma (CCA) is a highly lethal, heterogenous biliary malignancy. Despite the development of targeted therapies, treatment success has been hindered by primary or acquired resistance. New therapeutic strategies are necessary to improve outcomes in these patients. NXP800 is a GCN2 small-molecule activator that phosphorylates eIF2a inducing selective ATF4 transcription which stimulates stress-induced genes leading to apoptosis. NXP800 has been previously validated in ovarian cancers with ARID1A mutations, well-known players in cholangiocarcinoma. We sought to determine the treatment efficacy of novel NXP800 in CCA utilizing patient derived xenograft (PDX) preclinical models. Methods In our preliminary studies, four human and two murine CCA cell lines were tested for cell viability using CellTiter-Glo to determine the maximal inhibitory concentration (IC50). Immunoblot analysis of HuCCT-1 cells incubated with 1 mM of NXP800 for 6 hours vs. vehicle was performed. We also evaluated response to GCN2 activation in multiple RNA sequenced PDX models. Xenografts were expanded into the flank of NOD/SCID mice and treatment begun when tumors averaged 120mm3. Tumor bearing mice were treated with NXP800 (35 mg/kg) or vehicle via oral gavage five days on, two days off, for 28 days. Tumor volume and animal weight were collected twice weekly. Predicted sensitive signatures were determined using multi-omics. Results IC50 dose response curves showed nanomolar activity in all six cell lines, ranging from 5-170 nM. Immunoblotting demonstrated an upregulation of phosphorylated eIF2a and in turn upregulation of ATF4 with treatment of NXP800 compared to vehicle. Five PDX tumors from patients with intrahepatic or distal CCA was established and validated by histologic analysis. Molecular characterization of the tumor identified tumor mutation and a total tumor mutational burden. RNA sequencing was completed and identified 21-gene signatures related to YAP activity, which were cross-referenced against our existing PDX model tumor bank. Treatment with NXP800 was associated with a statistically significant decrease in tumor size in three of five PDX models tested, which included an ARID1A, FGFR1, and ATM mutants. Preliminary multi-omics on the PDX models revealed that sensitivity mechanisms were centered around EGFR signaling. Conclusion The novel GCN2 kinase activator, NXP800, has nanomolar efficacy in both human and murine CCA cell lines in vitro. Additionally, NXP800 demonstrated therapeutic activity in multiple cholangiocarcinoma PDX models in vivo. We are opening a Phase 1b clinical trial investigating the effects of NXP800 use in patients with advanced CCA. Future studies determining the cellular effect of GCN2 activation utilizing NXP800 are being conducted. Citation Format: Danielle Marie Carlson, Hendrien Kuipers, Amro Abdelrahman, Erik Jessen, Joshua Roldan Kalil, Jack Sample, Jennifer Tomlinson, Mark Truty, Rory Smoot. A novel GCN2 kinase activator demonstrates therapeutic efficacy in preclinical PDX models of human cholangiocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2098.

Decoding the Epigenetic Drivers of Menin-MLL Inhibitor Resistance in KMT2A-Rearranged Acute Myeloid Leukemia

Menin inhibitors that disrupt Menin-MLL interaction are candidate therapeutic agents for several acute myeloid leukemia (AML) subtypes such as KMT2A rearrangement and NPM1 mutation. Several Menin inhibitors are currently being assessed in clinical trials and have demonstrated promising initial results. However, the development of both innate and acquired resistance to Menin inhibitors could limit their therapeutic potential. While studies have found that around 40% of these resistance cases can be traced back to mutations in the Menin gene, the Menin gene remains unaltered in other majorities, suggesting the presence of other mechanisms driving resistance. Here, we developed a unique chromatin-focused CRISPR knockout library, named Advanced Catalogue of Epigenetic Regulators (ACER). The ACER library contains approximately 800 traditional and newly-predicted epigenetic regulators using epigenetic network analyses that integrated publicly available gene codependency (DepMap) and protein-protein interaction data (BioPlex) (Figure A). To understand the chromatin mechanisms responsible for Menin-MLL inhibition resistance, we employed the ACER library for CRISPR screen in the context of Menin inhibition. Notably, our screen identified non-canonical polycomb repressive complex PRC1.1 (PCGF1, BCOR, and RYBP) and PRC2.2 (EZH2, SUZ12, and EED) among the top hits that conferred resistance to MLL-Menin inhibition upon gene depletion (Figure B). Consistent with our screen results, the depletion of PRC1.1 components led to a markedly increase in IC50 values when treated with the Menin inhibitor VTP50469 in both human and murine KMT2A-rearranged cell models. While treatment with Menin inhibitors typically induces cellular differentiation in leukemia cells, depletion of PRC1.1 genes resulted in an almost complete blockage in this differentiation process. Menin inhibitors evict Menin from chromatin and repress the expression levels of Menin-MLL target genes like MEIS1, PBX3, and MEF2C. Strikingly, our ChIP-seq and RNA-seq data indicatethat the loss of PRC1.1 components doesn't impact Menin's displacement from chromatin or the repression of KMT2A target genes by the Menin inhibitor. This is consistent with recent data from phase I clinical trial of revumenib (SNDX-5613) and preclinical patient derived xenograft models, wherein the Menin inhibitor persistently inhibitedkey KMT2A targets, even in resistant samples. These findings highlightthe possibility that resistance to Menin inhibition arising from mechanisms independent of canonical KMT2A target genes. Importantly, our unbiased transcriptomic analysis revealed that MYC signatures as the most affected pathway in PRC1.1-depleted cells following Menin inhibition. Furthermore, we show that both the transcriptionally active Menin-MLL and the repressive PRC1.1 complexes coexist at the MYC promoter, and the interplay between these two complexes determines MYC transcription output in AML. Moreover, enforced expression of MYC leads to resistance against Menin inhibition, while genetic and pharmacologic targeting MYC in PRC1.1 deficient cells rescued the resistance to Menin inhibition. Finally, in our RNA-seq datasets, we observed a diminished monocyte differentiation signature after PRC1.1 depletion. Considering AML cells with monocytic signatures, typically seen in KMT2A-rearranged AML, often exhibit resistance to BCL-2 inhibitor venetoclax, we hypothesized that AML cells lacking PRC1.1 could be more susceptible to BCL2 inhibition. This was confirmed in our ACER library screen with venetoclax treatment, where the PRC1.1 components PCGF1 and BCOR stood out as top epigenetic factors rendering drug sensitivity. Our subsequent studies using murine AML models and human primary samples further validated that PRC1.1-deficient AML cells are markedly sensitive to venetoclax. Furthermore, we show that venetoclax can be employed to overcome the resistance to Menin-MLL inhibition. In summary, our study identifiesthe non-canonical PRC1.1 as a key epigenetic driver of Menin-MLL resistance through a KMT2A target gene-independent mechanism which involves aberrant activation of MYC. We have further provided evidence that AML cells with loss of PRC1.1 are hypersensitive to BCL-2 inhibitor Venetoclax, opening a new therapeutic avenue for tackling Menin-resistant AMLs driven by polycomb inactivation.

Radiosensitisation by olaparib through focused ultrasound delivery in a diffuse midline glioma model

Background and purposeDiffuse midline glioma H3K27-altered (DMG) is an aggressive, inoperable, predominantly paediatric brain tumour. Treatment strategies are limited, resulting in a median survival of only 11 months. Currently, radiotherapy (RT), often combined with temozolomide, is considered the standard of care but remains palliative, highlighting the urgency for new therapies. Radiosensitisation by olaparib, an inhibitor of PARP1 and subsequently PAR-synthesis, is a promising treatment option. We assessed whether PARP1 inhibition enhances radiosensitivity in vitro and in vivo following focused ultrasound mediated blood-brain barrier opening (FUS-BBBO). MethodsEffects of PARP1 inhibition were evaluated in vitro using viability, clonogenic, and neurosphere assays. In vivo olaparib extravasation and pharmacokinetic profiling following FUS-BBBO was measured by LC-MS/MS. Survival benefit of FUS-BBBO combined with olaparib and RT was assessed using a patient-derived xenograft (PDX) DMG mouse model. ResultsTreatment with olaparib in combination with radiation delayed tumour cell proliferation in vitro through the reduction of PAR. Prolonged exposure of low olaparib concentration was more efficient in delaying cell growth than short exposure of high concentration. FUS-BBBO increased olaparib bioavailability in the pons by 5.36-fold without observable adverse effects. A Cmax of 54.09 μM in blood and 1.39 μM in the pontine region was achieved following administration of 100 mg/kg olaparib. Although RT combined with FUS-BBBO mediated olaparib extravasation delayed local tumour growth, survival benefits were not observed in an in vivo DMG PDX model. ConclusionsOlaparib effectively radiosensitises DMG cells in vitro and reduces primary tumour growth in vivo when combined with RT. Further studies are needed to investigate the therapeutic benefit of olaparib in suitable preclinical PDX models.

Abstract 6553: The development of homologous recombination repair deficiency (HRD) score methodology using WES data and its application in patient-derived xenograft models

Abstract Homologous recombination repair deficiency (HRD) and mutations in genes involved with homologous recombination repair (HRR) are associated with sensitivity to platinum-based chemotherapy, topoisomerase 1 inhibitors and inhibitors targeting DNA repair pathway. Use of the Myriad HRD test for HRD determination in preclinical models is not feasible globally. To address this challenge, we developed an approach to generate HRD scores from whole exome sequencing (WES) data and applied it to assess HRD status in patient-derived xenograft (PDX) models. We used python package CNVkit for segmentation and gene-level/allele-specific copy number calling with customized settings according to CNVkit documentation. The reference was constructed using the WES BED file and hg38 reference genome when there are no paired control samples available. Next R package scarHRD was used to determine the HRD score that is the sum of loss of heterozygosity (LOH), telomere allele imbalances (TAI), and large fragment migrations (LST). The method was applied to the WES of 25 Xentech triple negative breast cancer (TNBC) PDX models to derive HRD scores. The samples were also separately profiled using Myriad’s MyChoice CDx assay. The two scores showed moderate correlation with Pearson r = 0.56. However, when n = 6 PDX models with low coverages (average depth <100x) were excluded, the Pearson r improved to r = 0.76. We also found that the absolute value of HRD score calculated by CNVkit/scarHRD is lower than that of Myriad HRD score. Furthermore, we evaluated the genomic landscape in this cohort of TNBC PDX models for the selected genes of HRR pathway (BRCA1, BRCA2, ATM, BRIP1, PALB2, RAD51C, BARD1, CDK12, CHEK1/2, FANCL, RAD51B, RAD51D, RAD54L). Sixteen models found to carry HRR mutations, with ATM and BRCA1 mutations being the most common ones, being found in 9 and 5 of 25 models, respectively. Although HRRm positive (HRRm+) models have higher HRD score for both Myriad HRD score and HRD score generated with CNVkit/scarHRD approach, the association between HRR (HRRm+/HRRm-) and Myriad HRD (HRD+/HRD- defined by HRD threshold of 42) is not statistically significant (Fisher’s exact test p value = 0.1998, odds ratio=3.5), likely due to small cohort sizes. These results suggest that combination of CNVkit and scarHRD for HRD score calculation may be an alternative approach for HRD identification in preclinical PDX models. Citation Format: Jixin Wang, Krista Kinneer, Wenyan Zhong, Zhongwu Lai. The development of homologous recombination repair deficiency (HRD) score methodology using WES data and its application in patient-derived xenograft models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6553.

Preclinical In Vivo Activity of the Menin Inhibitor Ziftomenib (KO-539) in Pediatric KMT2A-Rearranged Acute Lymphoblastic Leukemia

Background: Chemotherapy resistance and subsequent relapse remain a major cause of childhood cancer mortality, particularly for infants (<12 months of age) with KMT2A-rearranged (R) B-acute lymphoblastic leukemia (B-ALL), who have extremely poor clinical outcomes with event-free survival <20% for highest-risk patients. KMT2A (MLL1) is a histone lysine N-methyltransferase involved in HOX-family and MEIS1 gene regulation that is commonly rearranged with other gene partners to form oncogenic KMT2A fusion proteins that drive acute lymphoid or myeloid leukemogenesis. Recent preclinical and early clinical studies have reported successful targeting of the menin scaffold protein within KMT2A fusion complexes in adults with KMT2A-R acute leukemias (Wang ASH 2020 abstract #115, Stein ASH 2021 abstract #699), but minimal data in pediatric patients exist to date. We hypothesized that the selective menin inhibitor ziftomenib (KO-539) would have potent activity in preclinical patient-derived xenograft (PDX) models of infant and non-infant pediatric KMT2A-R ALL and could enhance chemosensitivity in these often-chemoresistant leukemias. Methods: To test these hypotheses, we first measured the ability of ziftomenib to inhibit in vitro viability and KMT2A-menin complex protein targets in human KMT2A-R and KMT2A wild-type ALL cell lines in vitro using Cell Titer Glo and immunoblotting assays, respectively. We then assessed the ability of ziftomenib as monotherapy or in combination with chemotherapy to inhibit in vivo leukemia proliferation in PDX models of de novo or relapsed KMT2A-R ALL harboring various fusion partners (n=8; Loftus Haematologica 2021) after drug dose-finding in a bioluminescent KMT2A-R ALL cell line xenograft model. NSG mice engrafted with human KMT2A-R ALL cells were randomized to treatment with vehicle, ziftomenib 150 mg/kg via oral gavage (PO) daily x 5 days/week, vincristine 1 mg/kg intraperitoneally once weekly, dexamethasone 1 mg/kg PO daily x 5 days/week, ziftomenib + vincristine, or ziftomenib + vincristine for 3-6 weeks depending on rate of ALL progression in control animals necessitating study termination. Human CD45+ CD19+ ALL cell counts were measured weekly via retro-orbital venous bleeding and in end-study murine spleens via quantitative flow cytometry analysis as previously described (Tasian Blood 2017a, Hurtz JCI 2020). Results: We observed preferential in vitro sensitivity to ziftomenib of KMT2A-R ALL cell lines HB11;19 (half-maximal inhibitory concentration [IC50] 11.1 nM), KOPN-8 (IC50 49.1 nM), and SEM (IC50 32.6 nM) versus KMT2A wild-type NALM-6 cells (IC50 400.8 nM). Ziftomenib treatment of infant, non-infant pediatric, and young adult KMT2A-R ALL PDX models (comprised of more common KMT2A-AFF1 and KMT2A-MLLT1 rearrangements or other less common KMT2A fusions) induced significant inhibition of in vivo leukemia proliferation compared to vehicle treatment. Despite the remarkable potency of single-agent ziftomenib in all models tested, combination of ziftomenib with vincristine or dexamethasone further enhanced reduction of human ALL disease burden in end-study murine spleens, even in aggressive relapsed infant ALL PDX models (representative data in Figure 1). Conclusions: Our data demonstrate in vitro target inhibition and remarkable in vivo activity of the menin inhibitor ziftomenib against infant, pediatric, and young adult KMT2A-R ALL. Ziftomenib monotherapy activity was further augmented in most models when combined with conventional chemotherapy drugs vincristine or dexamethasone, which were well-tolerated in PDX mice. Based upon early clinical safety/tolerability data for ziftomenib in adult patients with acute leukemias and our pediatric-specific data described herein, a phase 1 clinical trial of ziftomenib in combination with multi-agent chemotherapy for children with relapsed/refractory KMT2A-rearranged, NUP98-rearranged, or NPM1-mutant acute leukemias is planned. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Inhibition of sphingolipid metabolism in osteosarcoma protects against CD151-mediated tumorigenicity

Osteosarcoma is the most common primary bone tumor, with a poor prognosis owing to the lack of efficient molecular-based targeted therapies. Previous studies have suggested an association between CD151 and distinct consequences in osteosarcoma tumorigenicity. However, the potential of CD151 as a therapeutic target has not yet been sufficiently explored. Here, we performed integrated transcriptomic and metabolomic analyses of osteosarcoma and identified sphingolipid metabolism as the top CD151-regulated pathway. CD151 regulates sphingolipid metabolism primarily through SPTCL1, the first rate-limiting enzyme in sphingolipid biosynthesis. Mechanistically, depletion of CD151 enhanced c-myc polyubiquitination and subsequent degradation. c-myc is vital for the transcriptional activation of SPTLC1. Functionally, sphingolipid synthesis and the SPTLC1 inhibitor, myriocin, significantly suppressed the clonogenic growth of CD151-overexpression cells. Importantly, myriocin selectively restrained CD151-high expression tumor growth in preclinical patient-derived xenograft models. Collectively, these data establish that CD151 is a key mediator of sphingolipid metabolism and provide a new approach to developing novel CD151-based targeted therapies for osteosarcoma.

Longitudinal monitoring of disease burden and response using ctDNA from dried blood spots in xenograft models.

Whole‐genome sequencing (WGS) of circulating tumour DNA (ctDNA) is now a clinically important biomarker for predicting therapy response, disease burden and disease progression. However, the translation of ctDNA monitoring into vital preclinical PDX models has not been possible owing to low circulating blood volumes in small rodents. Here, we describe the longitudinal detection and monitoring of ctDNA from minute volumes of blood in PDX mice. We developed a xenograft Tumour Fraction (xTF) metric using shallow WGS of dried blood spots (DBS), and demonstrate its application to quantify disease burden, monitor treatment response and predict disease outcome in a preclinical study of PDX mice. Further, we show how our DBS‐based ctDNA assay can be used to detect gene‐specific copy number changes and examine the copy number landscape over time. Use of sequential DBS ctDNA assays could transform future trial designs in both mice and patients by enabling increased sampling and molecular monitoring.

Synergistic activity of combined inhibition of anti-apoptotic molecules in B-cell precursor ALL

Targeting BCL-2, a key regulator of survival in B-cell malignancies including precursor B-cell acute lymphoblastic leukemia, has become a promising treatment strategy. However, given the redundancy of anti-apoptotic BCL-2 family proteins (BCL-2, BCL-XL, MCL-1), single targeting may not be sufficient. When analyzing the effects of BH3-mimetics selectively targeting BCL-XL and MCL-1 alone or in combination with the BCL-2 inhibitor venetoclax, heterogeneous sensitivity to either of these inhibitors was found in ALL cell lines and in patient-derived xenografts. Interestingly, some venetoclax-resistant leukemias were sensitive to the MCL-1-selective antagonist S63845 and/or BCL-XL-selective A-1331852 suggesting functional mutual substitution. Consequently, co-inhibition of BCL-2 and MCL-1 or BCL-XL resulted in synergistic apoptosis induction. Functional analysis by BH3-profiling and analysis of protein complexes revealed that venetoclax-treated ALL cells are dependent on MCL-1 and BCL-XL, indicating that MCL-1 or BCL-XL provide an Achilles heel in BCL-2-inhibited cells. The effect of combining BCL-2 and MCL-1 inhibition by venetoclax and S63845 was evaluated in vivo and strongly enhanced anti-leukemia activity was found in a pre-clinical patient-derived xenograft model. Our study offers in-depth molecular analysis of mutual substitution of BCL-2 family proteins in acute lymphoblastic leukemia and provides targets for combination treatment in vivo and in ongoing clinical studies.

Therapeutic Effects of Dietary Soybean Genistein on Triple-Negative Breast Cancer via Regulation of Epigenetic Mechanisms

Consumption of dietary natural components such as genistein (GE) found in soy-rich sources is strongly associated with a lower risk of breast cancer. However, bioactive dietary component-based therapeutic strategies are largely understudied in breast cancer treatment. Our investigation sought to elucidate the potential mechanisms linking bioactive dietary GE to its breast cancer chemotherapeutic potential in a special subtype of aggressive breast cancer—triple-negative breast cancer (TNBC)—by utilizing two preclinical patient-derived xenograft (PDX) orthotopic mouse models: BCM-3204 and TM00091. Our study revealed that administration of GE resulted in a delay of tumor growth in both PDX models. With transcriptomics analyses in TNBC tumors isolated from BCM-3204 PDXs, we found that dietary soybean GE significantly influenced multiple tumor-regulated gene expressions. Further validation assessment of six candidate differentially expressed genes (DEGs)—Cd74, Lpl, Ifi44, Fzd9, Sat1 and Wwc1—demonstrated a similar trend at gene transcriptional and protein levels as observed in RNA-sequencing results. Mechanistically, GE treatment-induced Cd74 downregulation regulated the NF-κB/Bcl-xL/TAp63 signal pathway, which may contribute to soybean GE-mediated therapeutic effects on TNBC tumors. Additionally, our findings revealed that GE can modify expression levels of key epigenetic-associated genes such as DNA methyltransferases (Dnmt3b), ten-eleven translocation (Tet3) methylcytosine dioxygenases and histone deacetyltransferase (Hdac2), and their enzymatic activities as well as genomic DNA methylation and histone methylation (H3K9) levels. Collectively, our investigation shows high significance for potential development of a novel therapeutic approach by using bioactive soybean GE for TNBC patients who have few treatment options.

Synergistic Activity of IDH1 Inhibitor Bay-1436032 with Azacitidine in IDH1 Mutant Acute Myeloid Leukemia

Background: Only about one third of AML patients with mutant IDH1 respond to IDH1 inhibitor monotherapy with a median response duration of 7 months, indicating the strong need for combination treatments. We have previously shown that BAY-1436032 is a highly effective oral pan-mutant IDH1 inhibitor, which has strong anti-leukemic activity in patient derived xenograft (PDX) models of IDH1 mutant leukemia in vivo. In the present study, we assessed the combination of azacitidine with IDH1 inhibitor BAY-1436032 in a preclinical PDX model of IDH1 mutated AML.Methods: We tested the activity of BAY-1436032 alone or in combination with escalating doses of azacitidine in IDH1 mutated and IDH1 wildtype AML patient cells in vitro evaluating the effects on colony formation, apoptosis, cell cycle, differentiation and global gene expression changes. Leukemic cells from an AML patient with mutated IDH1, NPM1, FLT3-TKD and NRAS were xenografted in immunocompromised mice and treatment was started 28 days after transplantation. The control groups were treated with either vehicle, BAY-1436032 150 mg/kg once daily p.o. continuously, or azacitidine 1 mg/kg once daily s.c. days 1-5, repeated once after 28 days. The test groups were treated with BAY-1436032 and azacitidine in the doses mentioned above either starting both drugs on day 1 (parallel group) or starting azacitidine on day 1 but BAY-1436032 on day 6 (sequential group). The treatment was stopped after 84 days.Results: Our IDH1 inhibitor reduced colony formation specifically in human IDH1 mutated AML cells, while IDH1 wildtype cells were not affected (IC50 100 nM). Combination of BAY-1436032 (100 nM) with azacitidine 100 nM further reduced colony formation by 50% in IDH1 mutated AML cells, while the IC50 for IDH1 wildtype AML cells was not reached even at 1000 nM azacitidine in the presence of 100 nM BAY-1436032. For in vivo experiments human AML cells from an IDH1 mutated AML patient were transplanted into sublethally irradiated NSG mice and treatment was started four weeks after transplantation. Leukemic cells in peripheral blood constantly increased in vehicle and azacitidine treated mice, while leukemic cells declined from week 4 until the end of treatment at week 12 in mice treated with BAY-1436032 alone as well as in the parallel and sequential combination groups treated with BAY-1436032 and azacitidine. However, in week 20 (8 weeks after stopping the BAY-1436032 treatment) leukemia relapsed in the BAY-1436032 alone as well as the sequential combination groups. Interestingly, the frequency of leukemic cells remained low in peripheral blood in mice treated in parallel with the combination of BAY-1436032 and azacitidine even at 24 weeks after starting the treatment (Figure 1). To obtain mechanistic insights into the efficacy of the combination treatment we treated human IDH1 mutant and IDH1 wildtype AML cells in vitro with either 100 nm BAY-1436032 or 100 nm azacitidine alone or in combination (in parallel). Vehicle treatment served as control. There were no significant differences in percentage of apoptotic cells between BAY-1436032 or azacitidine treatment as single agents or in combination. However, the proportion of cells in S phase of the cell cycle was synergistically decreased by the combination treatment compared with either monotherapy or vehicle. Gene expression profiling showed that the target genes of EGR1-3 and GFI1 transcription factors were upregulated by the combination treatment over BAY-1436032 treatment alone, while target genes of NFkB and its targets ELF1 and STAT1/3 were down regulated, suggesting synergy in induction of differentiation and inhibition of self-renewal of the combination treatment through distinct pathways.Conclusion: Our study provides the first evidence of synergistic activity of an IDH1 inhibitor with hypomethylating agents through inhibition of the cell cycle by dysregulating EGR, GFI1 and NFkB signaling, and strongly argues for simultaneous application of the IDH1 inhibitor BAY-1436032 with azacitidine in future clinical trials. Clinical development is ongoing with phase 1 studies using BAY-1436032 in IDH1 mutant solid tumors and AML.Figure 1: Development of human IDH1 mutated AML in peripheral blood of NSG mice treated with BAY-1436032 and azacitidine alone or in combination (PRL, in parallel; SEQ, sequential, i.e. first azacitidine days 1-5, then BAY-1436032 from day 6 onwards). [Display omitted] DisclosuresChaturvedi:Bayer Pharma AG, Berlin, Germany: Research Funding. Kaulfuss:Bayer Pharma AG, Berlin, Germany: Employment. Panknin:Bayer Pharma AG, Berlin, Germany: Employment. Wagner:Bayer Pharma AG, Berlin, Germany: Employment, Equity Ownership. Jeffers:Bayer Pharma AG, Whippany, NJ, USA: Employment. Haegebarth:Bayer Pharma AG, Berlin, Germany: Employment, Equity Ownership.

Establishment of a Patient-Derived Acute B Lymphoblastic Leukemia (B-ALL)Ex Vivo and In Vivoplatform for Evaluation of Novel Therapeutic Agents

B-cell Acute Lymphoblastic Leukemia (B-ALL)is a hematological malignancy with malignant transformation and proliferation of B-Cell progenitors in bone marrow, blood and extramedullary sites. It is more prevalent in children and adolescent and is one of the top leading cause of death related to malignancies in this population. Majority of young patients fare well with common therapeutic agents and hematopoietic stem cell transplant, but a subpopulation is chemo-resistant and has a high relapse rate. Besides, prognosis in adult B-ALL patient is worse than younger individuals. Thus, there is an increasing need for development of novel and targeted therapeutic strategies to improve outcome in elderly and resistant individual. Lack of suitablein vitroculture conditions for primary B-ALL cells severely impairs their experimental accessibility and evaluation of new therapeutics. To corroborate theex vivostudies in a more natural microenvironment there is an unmet necessity of a robust preclinical patient derived xenograft model for B-ALL. We have established and optimized a short-term culture system that supports survival of primary B-ALL cellsex vivofor evaluation and/or screening of candidate agents. Our B-ALL bank comprise of patient-derived specimens with complete cytogenetics information including Philadelphia translocation positive and negative samples and surface marker expression. Different medias, supplements and culture conditions were evaluated to select conditions that support the survival and maintenance of primary B-ALL specimens. Cell growth/viability was assessed using Cell titer-Glo assay. Out of 13 primary B-ALL models studied 5 models proliferated in the optimized media and 5 remain stable and viable till day 3. Phenotypic characterization by flow cytometry showed that all the models stably expressed CD19 and CD20 throughout the assay. To verify the applicability of the 3-day ex vivo system for drug testing, models were tested for sensitivity towards chemotherapeutic drugs doxorubicin, vincristine, imatinib and cytarabine. Heterogeneous dose dependent responses were observed across the models (IC50 17nM to 0.3 µM doxorubicin), (IC50 3 nM to 79 nM vincristine) indicating a range of relative sensitivity to B-ALL models. Interestingly, BCR-ABL-ve models were resistant to imatinib and BCR-ABL+ve sensitive, when treated with small molecule inhibitor imatinib. Next, to develop patient derived xenograft preclinical B-ALL models for drug efficacy study , sub lethally irradiated NCG mice were transplanted intravenously with two million PBMC from B-ALL patients(n=14). 100% engraftment(n=5/model P1) was observed in all the models, but the latency of engraftment(&amp;gt;3%) in peripheral blood varied from 15 days to 3.5 months depending on the aggressiveness of the models. The time of passage varied from 40 to 120 days at P1. Mice were sacrificed when 10-20% of hCD45+ve cells were observed in peripheral blood by flow cytometry. 30-95% of the bone marrow was occupied by hCD45+, hCD19+ cells. Splenomegaly was observed and infiltration of 40-98% of B-ALL cells was detected in spleen. Engrafted cells were lymphoid-restricted and matched the immunophenotype of the corresponding patient. On serial transplantation in secondary NCG mice (n=15/model P2,P3), these models showed similar engraftment pattern and immunophenotype characteristic. Thus , here we show that theex vivoculture condition optimized at Champions supports growth and proliferation of patient derived B-ALL specimen and is a good platform for screening new therapeutic drugs for B-ALL . We also demonstrate that NCG mice supports the engraftment and development of B-ALL disease initiating and leukemic cells in vivo, allowing to create a robust passagable patient derived preclinical xenograft model that recapitulates the disease and probably could be used for drug efficacy study. Disclosures No relevant conflicts of interest to declare.

Establishment of a [18F]-FDG-PET/MRI Imaging Protocol for Gastric Cancer PDX as a Preclinical Research Tool.

PurposeThe utility of 18-fluordesoxyglucose positron emission tomography ([18F]-FDG-PET) combined with computer tomography or magnetic resonance imaging (MRI) in gastric cancer remains controversial and a rationale for patient selection is desired. This study aims to establish a preclinical patient-derived xenograft (PDX) based [18F]-FDG-PET/MRI protocol for gastric cancer and compare different PDX models regarding tumor growth and FDG uptake.Materials and MethodsFemale BALB/c nu/nu mice were implanted orthotopically and subcutaneously with gastric cancer PDX. [18F]-FDG-PET/MRI scanning protocol evaluation included different tumor sizes, FDG doses, scanning intervals, and organ-specific uptake. FDG avidity of similar PDX cases were compared between ortho- and heterotopic tumor implantation methods. Microscopic and immunohistochemical investigations were performed to confirm tumor growth and correlate the glycolysis markers glucose transporter 1 (GLUT1) and hexokinase 2 (HK2) with FDG uptake.ResultsOrgan-specific uptake analysis showed specific FDG avidity of the tumor tissue. Standard scanning protocol was determined to include 150 μCi FDG injection dose and scanning after one hour. Comparison of heterotopic and orthotopic implanted mice revealed a long growth interval for orthotopic models with a high uptake in similar PDX tissues. The H-score of GLUT1 and HK2 expression in tumor cells correlated with the measured maximal standardized uptake value values (GLUT1: Pearson r=0.743, P=0.009; HK2: Pearson r=0.605, P=0.049).ConclusionsThis preclinical gastric cancer PDX based [18F]-FDG-PET/MRI protocol reveals tumor specific FDG uptake and shows correlation to glucose metabolic proteins. Our findings provide a PET/MRI PDX model that can be applicable for translational gastric cancer research.

Targeted Inhibition of the NUP98-NSD1 Fusion Oncogene in AML

Background: NUP98-NSD1 positive AML is a poor prognostic subgroup within pediatric and adult AML (Thol et al., 2013). However, targeted therapeutics for these AML patients are not available to date. As a result of the NUP98-NSD1 fusion, NSD1 causes H3K36 hypermethylation of HOXA genes, which contributes to myeloid progenitor cell immortalization and results in AML (Wang et al., 2007). Therefore, we hypothesized that inhibition of the methyltransferase activity of NSD1 could be an effective treatment strategy for NUP98-NSD1 AML patients. Here, we assessed the efficacy of NSD1 inhibitor suramin and NUP98-NSD1-directed siRNA-containing lipid nanoparticles (LNP) in a preclinical patient-derived xenograft (PDX) model of NUP98-NSD1 leukemia. Methods: A NUP98-NSD1 positive AML patient was screened through nested PCR and Sanger sequencing. Bone marrow cells from this patient were serially transplanted into NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) mice to establish a NUP98-NSD1 PDX model. During serial transplantations, an alternative NUP98-NSD1 fusion gene appeared. Flow-cytometry was used to check the engraftment and immunophenotype of engrafted cells. Effective siRNAs against each of the two fusion genes were developed. The microfluidic mixing technology, Nanoassemblr was used to package siRNA into LNPs and the Zetasizer was used to characterize them. Fifteen days after transplantation, suramin and solvent treatment were initiated in 9 mice per group with 10 mg/kg suramin (2 days/week for 10 weeks). For LNP treatment, treatment was initiated 3 weeks after transplantation with a once daily dose of 2.5 mg/kg on days 1-3 and then every third day thereafter for a total of 17 injections. Results: Besides the NUP98-NSD1 translocation, our patient had a FLT3-ITD mutation and a normal karyotype. In our PDX model, the engraftment of human leukemia cells reached up to 90% after ten weeks of transplantation. In successive transplantations up to the 6th generation, the NUP98-NSD1 fusion was confirmed in leukemic cells, supporting the importance of the fusion for leukemia development and stability of the model. In later transplantations, a minor clone of NUP98-NSD1 was identified. A high blast count, high WBC count, increased spleen weight, and a low hemoglobin and platelet count at death demonstrated the development of acute leukemia. High expression of myeloid markers (e.g. CD33, 99%, N=9) and negligible expression of lymphoid markers (CD3, 2%; CD19, 2%; N=9) confirmed acute myeloid leukemia. In the suramin treatment study, the mean human leukemic cell engraftment was similar between the control and treatment groups at the start of treatment (0.51%, N=9 and 0.71%, N=9, respectively), but was lower in suramin treated mice after 4 and 8 weeks of treatment (4 weeks: CTRL, 4,8%; suramin, 2,66%, P=0.1; 8 weeks: CTRL, 87,3%; suramin: 66,5%, P=0.016). No significant effect was seen on the immunophenotype of suramin and control treated leukemia cells. Suramin treatment significantly prolonged the median survival of mice compared to control mice (126 vs 114 days after transplantation, P=0.008). To establish the siRNA-LNP treatment, we identified one siRNA against each NUP98-NSD1 clone that reduced expression levels by 78% and 89.5% in the major and minor clones, respectively. The effective siRNAs were modified to increase their in vivo stability and were packaged into LNPs and used in vivo. We started the treatment when the engraftment was similar in both control LNP and NUP98-NSD1 LNP groups (0.93%, N=7 and 1.25%, N=6, respectively). After 3 weeks of treatment, LNP uptake was 99.3% and 99.2% in the CTRL LNP and NUP98-NSD1 LNP groups, respectively. The mean engraftment was lower in NUP98-NSD1 LNP mice after 5 and 8 weeks of treatment (5 weeks: CTRL LNP, 15%; NUP98-NSD1 LNP, 4.6%, P= 0.08; 8 weeks: CTRL LNP, 94.8%; NUP98-NSD1 LNP, 55.83%, P=0.007). Importantly, the NUP98-NSD1 siRNA-LNP treated mice showed a significant survival benefit compared to CTRL siRNA-LNP treated mice (106 vs 82 days after transplantation, P=0.02). Conclusions: In summary, our findings demonstrate that targeted inhibition of NUP98-NSD1 either through siRNA-LNP or suramin delays leukemia development in vivo and prolongs the survival of mice carrying a NUP98-NSD1 positive AML. Our results provide the rationale for the evaluation of NSD1 methyltransferase inhibitors and siRNA-LNP formulations in NUP98-NSD1 positive AML. Disclosures Heuser: Bayer Pharma AG, Berlin: Research Funding; Synimmune: Research Funding.

Efficacy of Chemotherapy, Phd-Inhibitor Molidustat or BRD4 Inhibitor JQ1 in Combination with Targeted Inhibition of Mutated IDH1 in Human AML In Vivo

Background: Approximately 40% of AML patients with IDH1 mutation respond to monotherapy with the IDH1 inhibitor ivosidenib with a median duration of response of 8.2 months, suggesting that IDH1 inhibitors should be rationally combined with other agents to improve efficacy. We have previously shown the synergistic activity of the mutant IDH1 (mIDH1) inhibitor BAY1436032 with azacitidine. We have also shown that the leukemogenic activity of the mIDH1 protein depends on PHD3 independent of R-2HG and its inhibition as a novel therapeutic strategy (Chaturvedi et al. 2018). Inhibition of Brd4 has been shown to induce rapid differentiation and death of IDH2 mutated AML mouse models (Chen et al., 2013). In the present study, we assessed the combination of either conventional chemotherapy, prolyl hydroxylase (PHD) inhibitor molidustat or bromodomain inhibitor JQ1 with BAY1436032 (BAY) in a preclinical patient-derived xenograft (PDX) model of mIDH1 AML. Methods and Results: Leukemic cells from an AML patient with mutated IDH1, NPM1, FLT3-TKD and NRAS were xenografted in immunocompromised mice. We investigated the effects of BAY in sequential (seq) or simultaneous (sim) combination with cytarabine plus doxorubicin in our mIDH1 PDX model. The control groups were treated with either vehicle, BAY (150 mg/kg once daily p.o. continuously), or chemotherapy, which consisted of cytarabine (50 mg/kg once daily days 1-5 i.v.) and doxorubicin (1 mg/kg once daily days 1-3 i.v.). The treatment was repeated once after 29 days. The test groups were treated with BAY and chemotherapy in the doses mentioned above either starting both drugs on day 1 (sim group) or starting chemotherapy on day 1 and BAY on day 6 (seq group). Treatment with BAY was stopped after 12 weeks. Leukemic cells in peripheral blood constantly increased in vehicle and chemotherapy-treated mice with median time to 50% engraftment (MT 50%) of 84 and 112 days respectively. After stop of treatment, the percentage of leukemic cells increased in the group receiving BAY1436032 (MT 50%: 252 days) and sequential combination (MT 50%: 280 days), however, the MT50% was not reached with the simultaneous treatment (P&lt;0.001). Median survival for vehicle-treated mice was 173 days, 206 days for chemotherapy-treated mice, 325 days for BAY treated mice, and 340 days for the sequential combination treatment. Strikingly, 5/8 mice treated simultaneously with BAY and chemotherapy survived until the end of the study at 400 days and the median survival was not reached. We hypothesized that combination therapy with BAY and molidustat may demonstrate superior anti-leukemic activity in comparison to single agents in the treatment of IDH1-mutant AML. mIDH1 PDX mice were treated with either vehicle, BAY at a dose of 150 mg/kg or molidustat at a dose of 10 mg/kg p.o. as monotherapy or in combination. The MT50% for Vehicle, Molidustat and BAY was 70, 70 and 182 days respectively,. However, the MT50% was significantly delayed (322 days) for the combination treatment (P&lt;0.001). Interestingly, all mice treated with the combination had normal blood counts until week 40 and survived significantly longer than in the BAY group (median latency: 249 vs 179 days, P&lt;0.001). Mutant IDH1 PDX mice were treated with either vehicle, BAY, BRD4 inhibitor JQ1 (50mg/kg i.p. once daily) or the combination of BAY and JQ1 for 12 weeks. JQ1 monotherapy showed a significant delay in leukemia engraftment compared to vehicle-treated mice in the first 12 weeks of treatment. During the treatment, the leukemic cells remained low in both BAY and combination treated mice but relapsed at the same time 4 weeks after the treatment had been stopped. Median survival for vehicle-treated mice was 139 days, 164 days for JQ1-treated mice, 220 days for BAY treated mice, and 242 days for the combination treatment (BAY vs combination; P=0.03). Conclusion: Similar to the combination treatment of BAY with azacitidine the concurrent administration of BAY with chemotherapy significantly improves efficacy of this combination compared to sequential administration. In addition, the combination of an IDH1 inhibitor with molidustat is a promising therapeutic approach, while the combination with the BRD4 inhibitor JQ1 did not improve the outcome compared to treatment with an IDH1 inhibitor alone. Our findings support ongoing and future clinical investigations and suggest that IDH1 inhibitors should be applied concurrently with chemotherapy. Disclosures Chaturvedi: Bayer Pharma AG, Berlin, Germany: Research Funding. Kaulfuss:Bayer Pharma AG, Berlin, Germany: Employment. Panknin:Bayer Pharma AG, Berlin, Germany: Employment. Wagner:Bayer Pharma AG, Berlin, Germany: Employment, Equity Ownership. Jeffers:Bayer Pharma AG, Whippany, NJ, USA: Employment. Haegebarth:Bayer Pharma AG, Berlin, Germany: Employment, Equity Ownership. Heuser:Synimmune: Research Funding; Bayer Pharma AG, Berlin: Research Funding.

PF151 RAS/MAPK ACTIVATION COOPERATES WITH GAIN OF CHROMOSOME 21 IN B CELL LEUKEMIA AND IS AN ATTRACTIVE TARGET TO IMPROVE THE OUTCOME OF DS CHILDREN WITH B‐ALL

Background:B cell precursors acute lymphoblastic leukemia (B‐ALL) is the most commonly diagnosed childhood cancer. Major advances in treatments protocols, allowing for the 5‐year overall survival rate to reach 90%. However, many children still undergo intensive therapy and will develop relapse after a complete remission. Children with Down syndrome (DS) have a 27‐fold increased risk of developing B‐ALL and face a dismal prognosis due to treatment‐related mortality, and an increased risk of infections and relapse rate.Aims:The goal of the study was to better understand the molecular bases of DS‐ALL and develop specific therapies to improve outcomes.Methods:Here, we comprehensively characterized DS‐ALL samples, extended our study to other groups of childhood B‐ALL carrying gains of chromosome 21 (B‐ALL+21) and developed Patient Derived Xenograft (PDX) models suitable for preclinical studies. We performed exome and RNA sequencing to characterize the genetic and transcriptomic landscape of DS‐ALL (n = 8), iAMP21‐ALL (n = 7), HeH‐ALL (n = 14) compared to other pediatric B‐ALL (n = 12).Results:Somatic mutations leading to RAS/MAPK pathway activation (affecting the N/KRAS, JAK2, FLT3 and CBL genes) were present in 75% (22/29) of B‐ALL +21 samples, confirming recent observations by other group, and strengthening the link between RAS/MAPK pathway activation and +21. To gain insights into the molecular bases of this cooperation, we ectopically expressed the constitutively active mutant KRASG12D in murine bone marrow progenitors. We observed that KRASG12D expression in trisomic progenitors led to an increased number of CFU‐preB colonies that presented increased self‐renewal capacities compared to wild‐type, trisomy 21 or KRASG12D alone. RNA sequencing showed that 261 genes were synergistically deregulated by KRASG12D+Trisomie 21 in murine B cell progenitors, including 24 genes that were transcriptionally deregulated in B‐ALL+21 with RAS/MAPK mutations patient samples as compared to healthy B‐cell progenitors. Together, tshese results indicated that constitutive RAS activation and +21 functionally cooperates at the cellular and molecular level to impose an altered differentiation of B‐cell progenitors.From this cohort, we also developed 18 preclinical PDX models (13 B‐ALL+21 and 5 Others) to test the efficacy of inhibitors of the RAS/MAPK pathway. We showed that the most potent compound, Trametinib (GSK1120212), significantly decreased viability of B‐ALL+21 blasts presenting a constitutive RAS/MAPK activation in vitro (IC50 = 0,1 ± 0,25 μM compared to 3,95 ± 4,7 μM for B‐ALL samples without RAS/MAPK activation). In vivo treatments with Trametinib significantly decreased leukemia burden in the bone marrow and spleen compared to vehicle controls, and increased survival of DS‐ALL, iAMP21‐ALL and HeH‐ALL PDX models. Finally, we assessed the efficacy of Trametinib in combination with conventional chemotherapy agents. We observed that Trametinib synergised with Vincristine in vitro, and delayed leukemia progression in vivo to enhance survival of DS‐ALL PDXs.Summary/Conclusion:Altogether, we built a comprehensive cohort of preclinical models of DS‐ALL and B‐ALL+21, and suggested that inhibiting RAS/MAPK pathway activation in combination with current chemotherapy may represent a promising strategy to improve the outcome of Down syndrome children with B‐ALL.

Abstract P4-04-17: Elacestrant (RAD1901) demonstrates anti-tumor activity in a fulvestrant-resistant PDX model

Abstract Background: Breast cancer is subdivided into categories based on tumor receptor status, with estrogen-receptor positive (ER+) breast cancer making up approximately two-thirds of all breast cancers diagnosed. In advanced or metastatic disease, dependence on ER signaling is often retained even after multiple rounds of endocrine therapy, supporting the use of ER targeting agents for several lines of treatment prior to chemotherapy. Selective estrogen receptor degraders (SERDs) have gained recent attention as ER antagonists given their ability to target and degrade ER. Fulvestrant, the only approved SERD on the market, is currently used as a second-line therapy in the metastatic setting, however, the intramuscular route of administration and pharmacokinetic properties of fulvestrant have fueled the development of more potent and orally bioavailable SERDs. We have previously described elacestrant (RAD1901), a novel and orally bioavailable SERD, as an inhibitor of ER+ breast cancer growth in preclinical patient-derived xenograft (PDX) models, including those that are insensitive to fulvestrant. Here, we describe elacestrant activity in an ER+ PDX model that had been treated with multiple rounds of fulvestrant treatment. Methods: An ER+/PR+ PDX model, MAXF-713, derived from a treatment-naïve patient, was passaged multiple times in the presence of fulvestrant over the course of a year. After each round of fulvestrant treatment, elacestrant or fulvestrant were evaluated for anti-tumor activity. Pharmacodynamic endpoints including changes in ER and ER target gene expression were also evaluated. Results: Despite repeated exposure to fulvestrant, the PDX model retained sensitivity to elacestrant. In fact, regardless of the number of prior rounds of fulvestrant treatment, elacestrant produced consistent tumor growth inhibition within every passage, while the effects produced by fulvestrant exhibited a high degree of variability. Conclusions: Our data demonstrate that elacestrant is a SERD that can inhibit tumor growth in a fulvestrant-resistant preclinical setting and provides rationale for examining elacestrant in the clinical setting in patients that have progressed on fulvestrant treatment. Citation Format: Arlt H, Garner F, Bihani T. Elacestrant (RAD1901) demonstrates anti-tumor activity in a fulvestrant-resistant PDX model [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P4-04-17.

PATIENT-DERIVED XENOGRAFTS AS A PRECLINICAL MODEL FOR BONE SARCOMAS.

ABSTRACTObjective:The purpose of this study was to reproduce a mouse model of bone sarcomas for use in cancer research. Methods:A fresh sample of the tumor tissue was implanted subcutaneously into nude mice. When the patient-derived xenograft (PDX) reached a volume of 1500 mm3, it was harvested for re-implantation into additional mice. Histology was used to compare the morphological characteristics of different generations of sarcoma xenografts with the primary tumor. Results:Sixteen sarcoma tissue samples were engrafted into nude mice. Nine patients were diagnosed with osteosarcoma, two with chondrosarcoma, two with malignant peripheral nerve sheath tumor, one with synovial sarcoma, one with pleomorphic sarcoma, and one with Ewing’s sarcoma. PDX tumors were generated in 11 of the 16 implanted specimens (69% success rate in P1). Six P1 tumors grew sufficiently for transfer into additional mice, producing the P2 generation, and three P2 tumors established the P3 generation. Conclusion:PDX tumors generated from bone sarcomas were successfully established in immunodeficient mice and reproduced the characteristics of the primary tumor with a high degree of fidelity. The preclinical PDX model described herein may represent an important tool for translational oncology research and for evaluating therapeutic strategies for bone sarcomas. Level of Evidence I; Experimental study.

Safety and efficacy of the BCL2 inhibitor venetoclax in estrogen receptor (ER) and BCL2-positive metastatic breast cancer: The mBEP study.

1044 Background: The anti-apoptotic protein BCL2 is overexpressed in ~85% of ER+ breast cancer (BC). Venetoclax (ABT-199), a BCL2 inhibitor approved for CLL (400 mg/day), synergizes with tamoxifen in preclinical patient derived xenograft models by increasing apoptosis. In the first study to evaluate venetoclax in solid tumors, we tested the safety and efficacy of this combination in ER+BCL2+ metastatic BC. Methods: A ‘3+3 design’ dose escalation phase 1b study enrolled women with ER+ ( &gt; 1%), BCL2+ ( &gt; 10%, mod-high) and HER2 non-amplified metastatic BC. Patients received escalating doses of venetoclax 200, 400, 600 or 800 mg/day with tamoxifen 20 mg/day. The primary objective was to determine dose-limiting toxicity (DLT) and maximum tolerated dose (MTD) over 4 weeks. There was no limit to the number of prior lines of therapy. Results: Fifteen patients were enrolled (mean age 62 years, range 44-78; previous tamoxifen, 10 pts). Mean lines of prior therapy for metastatic BC was 2.5 (median 2, range 0-6) and included tamoxifen (6 pts). ESR1 mutations were present in ctDNA of 4 patients. Treatment was well tolerated, with no DLT observed. MTD was not reached; 6 patients received the maximal planned dose (800 mg). The most common adverse event (AE) was lymphopenia (67% Grade 1-2; 13% Grade 3; No Grade 4), followed by nausea (46%, Grade 1-2), which was readily managed. Of 13 women with measurable disease (RECIST v1.1), 4 (31%) had a partial response and 5 (38%) had stable disease (clinical benefit rate, 69%). For patients with a partial response, tumor regression was rapid (evident at first restaging) and occurred in the 400-800 mg dose levels. Two patients with non-measurable bone-only disease had clinically stable disease (1 ongoing &gt; 64 weeks). The median duration of response has not yet been reached (range, 12 to &gt; 64 weeks). Conclusions: We demonstrated the safety of tamoxifen and venetoclax in ER+BCL2+ metastatic BC, with preliminary evidence of clinically relevant activity. A dose expansion study including serial biopsy, ctDNA and PET scans is ongoing at the 800 mg/day recommended phase 2 dose. Sponsor: Royal Melbourne Hospital Clinical trial information: ISRCTN98335443 , ACTRN12615000702516.

Abstract P4-21-18: Pre-clinical antitumor activity, tumor localization, and pharmacokinetics of MP0274, an apoptosis inducing, biparatopic HER2-targeting DARPin®

Abstract Background: HER2 positivity is an important predictive factor for treatment with anti-HER2 agents in several cancers. However, currently available monoclonal antibody and tyrosine kinase inhibitor drugs rarely achieve full disease control. We have developed a new HER2-targeting molecule with a unique pro-apoptotic mode of action that may provide additional benefit to patients. The DARPin® MP0274* binds to two distinct non-overlapping HER2 epitopes and to human serum albumin for half-life extension. As previously shown**, in vitro, MP0274 induces apoptosis and inhibits proliferation of cells expressing HER2 (IHC3+, IHC2+ and IHC1+) and potently inhibits HER2/HER3 downstream signaling. To support clinical development of MP0274, we tested the potency of MP0274 in several HER2 expressing patient-derived xenograft (PDX) models and investigated tumor localization. In addition, pharmacokinetics (PK) analysis was performed in cynomolgus monkeys. Methods: Antitumor activity of MP0274 was tested in breast and gastric HER2 expressing PDX mouse models and was compared to standard of care therapies. Tumor localization of MP0274 was studied using an Indium-111 labeled version of MP0274 in a human ovarian adenocarcinoma (SKOV-3) xenograft model by whole-body SPECT/CT imaging. The PK of MP0274 was studied in cynomolgus monkeys (MP0274 is cross-reactive with cynomolgus HER2). Results: In breast and gastric cancer PDX models, MP0274 showed superior efficacy compared to trastuzumab and lapatinib and equivalent efficacy compared to trastuzumab plus pertuzumab as measured by relative tumor volume. The imaging study with SPECT/CT demonstrated that MP0274 localizes effectively to the HER2-expressing human tumor within 24 h. The PK study in cynomolgus monkeys showed a half-life of ≥5 days at doses of 5 and 10 mg/kg while at the lowest dose tested (1 mg/kg) MP0274 had a terminal half-life of 0.4 days. The PK results are indicative of target-mediated clearance that becomes saturated at doses above 1 mg/kg. Conclusions: MP0274, with its unique pro-apoptotic mode of action, demonstrates excellent activity in preclinical PDX models, fast localization to tumor and a long half-life in cynomolgus monkeys. MP0274 was well tolerated in all studies. These results suggest that MP0274 has the potential to provide additional clinical benefit to patients with HER2-expressing tumors. A GLP repeated dose toxicology study is ongoing and a phase I clinical trial is in preparation. * DARPins are small repeat proteins, designed to bind targets with high affinity and specificity, which can be combined in a modular fashion to produce multi-functional agents. ** U. Fiedler et al. SABC 2013. Abstract# 1094 &amp; Poster# P4-12-30. Citation Format: Fiedler U, Metz C, Zitt C, Bessey R, Béhé M, Blanc A, Schibli R, Dolado I, Herbst J, Dawson KM, Kiemle-Kallee J. Pre-clinical antitumor activity, tumor localization, and pharmacokinetics of MP0274, an apoptosis inducing, biparatopic HER2-targeting DARPin® [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P4-21-18.