Citations

Abstract Lisavanbulin (LIS; BAL101553) is the prodrug of BAL27862, a microtubule-binding, tumor checkpoint controller and potential radiosensitizer. These studies evaluated optimal integration of LIS with standard of care radiation therapy (RT) and/or temozolomide (TMZ) using GBM PDX models. Distribution across the blood brain barrier was evaluated after a single 30 mg/kg oral LIS dose, and concentrations of the active metabolite BAL27862 were measured by liquid chromatography-tandem mass spectrometry. Similar BAL27862 concentrations were detected in the brain (B) and plasma (P) at both two (B:P ratio 1.29) and six hours (B:P ratio 1.64) post-dose. An in vivo screen of LIS monotherapy across 14 orthotopic GBM PDX models showed significant survival benefit (p<0.01) in seven models (median survival extension 24-87%). Extending from these results, LIS was evaluated in several of the sensitive models in combination with RT +/- TMZ. Two MGMT unmethylated PDXs, GBM6 and GBM150, were treated with vehicle or two weeks of RT +/- LIS. LIS dosing during the RT dosing period did not significantly improve median survival in either line (GBM6 survival with RT 54 days vs RT/LIS 58 days, p=0.16; GBM150 RT 86 days vs RT/LIS 101 days, p=0.21). However, prolonged LIS dosing from the start of RT until mice reached a moribund state demonstrated added benefit (GBM6 median 90 days vs RT 69 days, p=0.0001; GBM150 median 143 days vs RT 73 days, p=0.06). In GBM6, prolonged LIS dosing also significantly extended survival when combined with 2 weeks of RT/TMZ (median 101 days vs 66 days, p<0.0001), while LIS alone or RT/TMZ resulted in similar median survivals (63 days vs 66 days, respectively; p=0.68). This same RT/TMZ/LIS benefit was not seen in the MGMT methylated GBM12. Subsequent experiments were performed to evaluate integration of prolonged LIS dosing with concurrent RT/TMZ followed by 3 cycles of adjuvant TMZ (‘Stupp’ regimen). In MGMT methylated GBM39, LIS alone did not significantly extend survival, but LIS addition to the Stupp regimen doubled median survival (Stupp 249 days vs Stupp/LIS 502 days, p=0.0001). GBM150 demonstrated equal benefit from LIS alone or Stupp regimen (median 118 days vs 123 days, p=0.49). Stupp/LIS showed no additional survival benefit (median 98 days, p=0.97). In a second MGMT unmethylated, TMZ-resistant GBM26 PDX, LIS alone or combined with the Stupp regimen provided significant survival benefit: median survival 53 days for vehicle vs. 80 days for LIS (p=0.0001), 114 days for RT only (p<0.0001), 147 days for RT/LIS (p=0.30 relative to RT), 121 days for ‘Stupp’ regimen alone (p=0.57 relative to RT), and 172 days for Stupp/LIS (p=0.04 relative to Stupp). A follow-up GBM39 study revealed a significant increase in the mitotic marker phospho-histone H3 with LIS treatment relative to vehicle-treated controls (p=0.01) while Ki67 levels were similar (p=0.15). This suggests that LIS induces a mitotic arrest associated with microtubule deregulation. Collectively, these data provide a strong rationale to evaluate lisavanbulin (BAL101553) with RT +/- TMZ in GBM and provided the basis for an ongoing Phase I clinical trial. Citation Format: Danielle M Burgenske, Ann C Mladek, Jenny L Pokorny, Heidi A Lane, Felix Bachmann, Rachael A Vaubel, Mark A Schroeder, Katrina K Bakken, Lihong He, Zeng Hu, Brett L Carlson, Surabhi Talele, Gautham Gampa, Matthew L Kosel, Paul A Decker, Jeanette E Eckel-Passow, William F Elmquist, Jann Sarkaria. Modeling the clinical paradigm of lisavanbulin (BAL101553) deployment in patient-derived xenografts (PDX) of glioblastoma (GBM) [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 C096. doi:10.1158/1535-7163.TARG-19-C096

Abstract Cell survival following radiation therapy (RT) is critically modulated by multiple DNA damage response pathways including the p53 tumor suppressor, which promotes cell cycle arrest and/or apoptosis in the face of DNA damage. Murine Double Minute 2 (MDM2) targets p53 for degradation and suppresses its functions. The MDM2 locus is amplified in approximately 14% of glioblastoma (GBM) tumors and is a promising target for individualized therapy. In these studies, we tested the MDM2 small molecule inhibitor KRT-232, alone and in combination with RT, in MDM2-amplified and/or p53 wildtype patient-derived xenograft (PDX) models of GBM in vitro and in vivo. KRT-232 suppressed GBM PDX cell explant culture viability in three MDM2-amplified lines (GBM46, 108, and 148) and two non-amplified but p53 wildtype lines (GBM10 and 39) with similar IC50s ranging from 300-800nM in FBS culture conditions. As expected, little suppression was observed in a p53 mutant (GBM43) line. Many drugs have limited distribution in the brain, and to understand limitations surrounding KRT-232, LCMS-MS was used to compare brain and plasma levels of drug in mice. Preliminary pharmacokinetic analysis using FVB wildtype mice compared to mice which maintain triple knockout of the efflux transporters Mdr1a, Mdr1b and Bcrp1–/– (TKO) treated with a single 10 mg/kg oral dose of KRT-232 and harvested 2 hours later demonstrate an 8% brain/plasma ratio of KRT-232 in the TKO mice, while levels were undetectable in WT mice. In light of these data, the efficacy of KRT-232 was initially tested in vivo in an MDM2-amplified GBM108 PDX with an artificially disrupted BBB afforded by VEGFA lentiviral expression. In this model, daily dosing with KRT-232 alone produced a 100 day prolongation in survival as compared to vehicle. In a subsequent experiment, just one week of daily KRT-232 dosing, either alone or in combination with RT, in the control GBM108 PDX with a relatively intact BBB, was remarkably effective; median survival for placebo 22 days, KRT-232 alone 46 days, RT alone 31 days, and the KRT-232/RT combination 77 days. Despite low BBB penetration as determined by pharmacokinetic studies, KRT-232 alone and in combination with RT is an effective treatment in a pre-clinical model of glioblastoma. Future studies will evaluate KRT-232 pharmacodynamic responses as well as PDX orthotopic combination therapy in additional MDM2 amplified and non-amplified/p53 wildtype lines to understand if this therapy can be used in a larger population of GBM tumors. Citation Format: Ann C Mladek, Shiv Gupta, Minjee Kim, Afroz Shareef Mohammad, Katrina K Bakken, Helen He, Zeng Hu, Danielle M Burgenske, Brett L Carlson, William F Elmquist, Jann Sarkaria. MDM2 inhibitor KRT-232 extends survival in glioblastoma patient-derived xenograft models [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 C051. doi:10.1158/1535-7163.TARG-19-C051

Abstract Implanted neural stimulation and recording devices hold vast potential to treat a variety of neurological conditions, but the invasiveness, complexity, and cost of the implantation procedure greatly reduce access to an otherwise promising therapeutic approach. To address this need, a novel electrode that begins as an uncured, flowable prepolymer that can be injected around a neuroanatomical target to minimize surgical manipulation is developed. Referred to as the Injectrode, the electrode conforms to target structures forming an electrically conductive interface which is orders of magnitude less stiff than conventional neuromodulation electrodes. To validate the Injectrode, detailed electrochemical and microscopy characterization of its material properties is performed and the feasibility of using it to stimulate the nervous system electrically in rats and swine is validated. The silicone‐metal‐particle composite performs very similarly to pure wire of the same metal (silver) in all measures, including exhibiting a favorable cathodic charge storage capacity (CSC C ) and charge injection limits compared to the clinical LivaNova stimulation electrode and silver wire electrodes. By virtue of its simplicity, the Injectrode has the potential to be less invasive, more robust, and more cost‐effective than traditional electrode designs, which could increase the adoption of neuromodulation therapies for existing and new indications.

Abstract Through the recent elucidation of molecular and cellular processes in multiple myeloma (MM) pathogenesis that effect well-defined proliferative and survival pathways, a number of molecular targets for anti-cancer agents have emerged. This includes proteins involved in cell cycle regulation. A recent model of early MM pathogenesis proposes that at least one of the cyclin D genes is dysregulated in all myeloma tumors facilitating activation of cyclin dependent kinase (CDK)4 (or CDK6), and transition from G1 to S phase. It is hypothesized that this renders myeloma cells more responsive to bone marrow (BM) derived proliferative stimuli including IL-6 that upregulates MYC expression in MM cells and cooperates with cyclin D to promote transit to S-phase. In addition, recent studies have established a causative role of MYC dysreguation in progression from monoclonal gammopathy to myeloma. Thus an approach targeting deregulated cell cycle progression and MYC may prove effective MM therapy. Purvalanol is a potent and selective inhibitor of CDK1, an important regulator of cell cycle progression and has been reported to induce MYC-dependent apoptosis. Activity of purvalanol against a panel of 14 standardized, annotated myeloma cell lines was measured in a 48 hour MTT viability assay. IC50-s of all 14 cell lines ranged between 5 uM to 7.5 uM. Western blot and real-time PCR analysis revealed variability of MYC protein and mRNA levels between the 14 myeloma cell lines. We compared potential therapeutic activity of purvalanol against 3 myeloma cell lines (U266, H929, KMS12PE) with low, intermediate, and high levels of c-MYC expression, respectively. All three cell lines demonstrated G2-M growth arrest however marked apoptosis as determined by PI/annexin V staining was observed only in the cell lines with the highest level of MYC expression. Exposure of MM patient-derived BM mononuclear cells to purvalanol preferentially induced apoptosis of CD138+ MM cells. In contrast, purvalanol was minimally cytotoxic to the non-myeloma cell fraction and to non-transformed fibroblast cell lines (MRC5, IMR90 and W138) and failed to inhibit normal bone marrow-derived CD34 colony formation. Recent studies have demonstrated that the BM microenvironment offers protection of myeloma cells from chemotherapeutic agents by common mechanisms. Myeloma cell lines were cultured in 3 different conditions to mimic the tumor’s protective microenvironment. Soluble factors produced by the BM, IL6 and IGF-1 induced a modest degree of resistance to purvalanol while co-culture on BM stroma cells was completely protective. Studies evaluating the in vivo efficacy of purvalanol in a novel Vk*myc transgenic mouse model that spontaneously develops myeloma with a low proliferative index are ongoing and will be presented. The CDK1 inhibitor, purvalanol demonstrates broad single agent activity against myeloma cells with enhanced activity against MYC overexpressing cells. However, the strong protective effect of the BM microenvironment suggest that combination with agents that can reverse cell-adhesion mediated drug resistance may prove beneficial in optimizing the efficacy of this class of drugs for the treatment of MM.

Abstract Background: Peripheral T-cell lymphomas (PTCLs) comprise a heterogeneous group of relatively uncommon and generally aggressive non-Hodgkin lymphomas. Recent studies have begun to reveal their mutational landscape, such as the recurrent mutations affecting RHOA, IDH2, and TET2 in angioimmunoblastic T-cell lymphomas (AITLs) and related PTCLs. Here, we studied the exomes of 63 PTCLs to address several unanswered questions: (1) How frequently do PTCLs have mutations with a strong rationale for being actionable using clinically available targeted therapies? (2) What pathways are enriched in PTCLs that could point to additional targeted therapies? (3) What additional recurrent mutations exist that may provide insight into PTCL pathogenesis and classification? Methods: We performed exome capture and sequencing in 63 frozen PTCL samples (with matched germline controls from 22), including 13 PTCLs, not otherwise specified; 6 AITLs; 37 anaplastic large cell lymphomas (ALCLs: 12 ALK+, 19 ALK-, and 6 cutaneous); 4 extranodal NK/T-cell lymphomas; 2 cutaneous T-cell lymphomas, and 1 enteropathy associated T-cell lymphoma. Following primary variant calling, data were filtered by removing variants that were: outside the capture region, polymorphic (0.1% in ExAC), seen in paired germline or 50 healthy controls, within repetitive elements, or synonymous. Potentially actionable variants required published evidence for altering protein function and demonstrated in vivo or in vitro sensitivity to an available targeted agent (including open clinical trials). Mutated cancer genes (COSMIC) were examined for enrichment of KEGG canonical signaling pathways. A recurrent mutation encoding MSC E116K was validated in tumor DNA and MSC protein was examined by immunohistochemistry. Results: Of 25 unique variants with published evidence suggesting actionability (including ErbB, JAK/STAT, and RAS genes), at least one variant was present in 16/63 patients (25%). Variants in any COSMIC gene were seen in 55/63 samples (87%). Top genes were STAT3, RHOA, ARID1A, MLL2 (KMT2D), PTPRB, TP53, and TET2. The KEGG ErbB pathway was most significantly enriched for mutated genes in our dataset (EGFR, ERBB2, ERBB3, ERBB4, and others; q=3.88x10-3), followed by neurotrophin and mitogen-activated protein kinase signaling pathways. Among 25 recurrent non-synonymous variants, we identified MSC E116K with predicted gain-of-function exclusively in 3 ALK-ALCLs and 1 cutaneous ALCL. All mutated cases strongly expressed MSC protein. Among 96 cases, 13/18 ALK-ALCLs (72%) were positive for MSC, compared to 9/78 (12%) from other PTCL subtypes. Conclusions: Mutations that were potentially actionable based on published variant-specific data were identified in 25% of PTCLs, indicating promise for the role of sequencing in individualizing therapy. Additionally, remaining variants were enriched for several targetable pathways, most significantly ErbB. A novel recurrent mutation, MSC E116K, was seen exclusively in ALK-ALCLs (systemic and cutaneous). MSC protein, a repressor of E2A-mediated transcription of targets such as MYC, was expressed in most ALK- ALCLs and generally was absent in other PTCL subtypes. This finding extends evidence for a genetic relationship between systemic and cutaneous ALCLs and suggests MSC may help distinguish ALK- ALCLs from other PTCLs. Disclosures Link: Genentech: Consultancy, Research Funding; Kite Pharma: Research Funding.

AbstractAnaplastic large cell lymphomas (ALCLs) represent a relatively common group of T-cell non-Hodgkin lymphomas (T-NHLs) that are unified by similar pathologic features but demonstrate marked genetic heterogeneity. ALCLs are broadly classified as being anaplastic lymphoma kinase (ALK)+ or ALK−, based on the presence or absence of ALK rearrangements. Exome sequencing of 62 T-NHLs identified a previously unreported recurrent mutation in the musculin gene, MSCE116K, exclusively in ALK− ALCLs. Additional sequencing for a total of 238 T-NHLs confirmed the specificity of MSCE116K for ALK− ALCL and further demonstrated that 14 of 15 mutated cases (93%) had coexisting DUSP22 rearrangements. Musculin is a basic helix-loop-helix (bHLH) transcription factor that heterodimerizes with other bHLH proteins to regulate lymphocyte development. The E116K mutation localized to the DNA binding domain of musculin and permitted formation of musculin–bHLH heterodimers but prevented their binding to authentic target sequence. Functional analysis showed MSCE116K acted in a dominant-negative fashion, reversing wild-type musculin-induced repression of MYC and cell cycle inhibition. Chromatin immunoprecipitation–sequencing and transcriptome analysis identified the cell cycle regulatory gene E2F2 as a direct transcriptional target of musculin. MSCE116K reversed E2F2-induced cell cycle arrest and promoted expression of the CD30–IRF4–MYC axis, whereas its expression was reciprocally induced by binding of IRF4 to the MSC promoter. Finally, ALCL cells expressing MSCE116K were preferentially targeted by the BET inhibitor JQ1. These findings identify a novel recurrent MSC mutation as a key driver of the CD30–IRF4–MYC axis and cell cycle progression in a unique subset of ALCLs.

Immune cell profiling identifies intratumoral T cell subsets associated with patient survival in follicular lymphoma.