Tumor Checkpoint Research Articles

Abstract LB-151: The novel tubulin-binding, tumor checkpoint controller BAL101553 has differential effects on tumor vascularization with IV and oral dosing and provides superior anti-tumor activity in combination with bevacizumab

Abstract Background: BAL101553 is a highly soluble prodrug of BAL27862, a novel, small molecule, microtubule-depolymerizer inducing tumor cell death through the spindle assembly checkpoint. BAL101553 has anti-cancer activity in diverse tumor models refractory to standard therapies, and is currently undergoing Phase 1/2a evaluation in advanced cancer patients with IV (weekly) and oral (daily) dosing schedules. Here, the vascular disruption activity of BAL101553 was evaluated in the context of administration route and implications for combinations with VEGF-targeted therapies. Methods: MTD IV (day[d] 1) and oral (d1 & d1 - 4) dosing regimens were tested in H460 NSCLC xenografts. Tumors were analyzed by IHC for proliferation (Ki67) and vascularization (CD31, lectin perfusion). Drug combinations were performed in Pgp-overexpressing, colon carcinoma SW480 xenografts using BAL101553 IV 1xweek (21.3 mg/kg) or oral daily (15 mg/kg) and bevacizumab IP d1 & 4 (5 mg/kg). Results: H460 tumor-bearing mice received once weekly (IV 25 mg/kg; oral 60 mg/kg; high Cmax regimens) or daily (oral 25 mg/kg; low Cmax, high AUC) BAL101553 regimens which elicited equipotent anti-tumor effects (ΔT/Cs = 26-34%). Although all treatments elicited a similar anti-proliferative response (% reduction of mean Ki67 signal+/-SD=10+/-1.2; p<0.05 vs. controls), tumor vessel density was most profoundly reduced by high Cmax regimens. Interestingly, daily-oral treatment (low Cmax, high AUC) had reduced tumor anti-vascular activity, while presenting equivalent anti-proliferative and anti-tumor efficacy. In vitro assays against tumor cell lines showed that sub-cytotoxic BAL27862 concentrations reduced HIF-1α protein levels in normoxic and hypoxic conditions, diminishing VEGF-expression and secretion, indicating direct interference of the drug on hypoxic adaptation and endothelial tube formation. Based on these data, combination of the anti-VEGF antibody bevacizumab with BAL101553 was assessed in the paclitaxel refractory SW480 tumor model, using MTD BAL101553 dosing (IV weekly & oral daily). All monotherapies induced an anti-tumor response (ΔT/Cs bevacizumab: 35%; IV BAL101553 22%; oral BAL101553: 53%). However, bevacizumab combined with IV or oral BAL101553 was superior to single agents (ΔT/C: 6% and 17%, resp., p<0.05 vs. control) with IV combination associated with the least functional tumor vasculature. Conclusions: BAL101553 targets tumor cell proliferation, tumor hypoxic adaptation and vascularization. The latter is Cmax-driven and can be attenuated by daily oral dosing, providing an alternative pharmacodynamic endpoint with implications for clinical treatment strategies. IV and orally administered BAL101553 both elicit superior anti-tumor responses in combination with anti-VEGF therapy. Citation Format: Ashish Sharma, Felix Bachmann, Angela Broggini-Tenzer, Matthias Guckenberger, Heidi Lane, Martin N. Pruschy. The novel tubulin-binding, tumor checkpoint controller BAL101553 has differential effects on tumor vascularization with IV and oral dosing and provides superior anti-tumor activity in combination with bevacizumab [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-151. doi:10.1158/1538-7445.AM2017-LB-151

A phase I study to assess the safety, pharmacokinetics (PK), pharmacodynamics (PD) and antitumor activities of daily oral BAL101553, a novel tumor checkpoint controller (TCC) in adult patients with progressive or recurrent glioblastoma (GBM) or high-grade glioma.

TPS2601 Background: BAL101553 (prodrug of BAL27862) is a novel TCC that promotes tumor cell death by modulating the spindle assembly checkpoint. BAL27862 is a lipophilic, small molecule (MW 387) shown in rats to penetrate the brain (1:1 plasma ratio) and has shown promising antitumor activity in orthotopic preclinical models of GBM as monotherapy or in combination with radiotherapy (RT) with/without chemotherapy. In a completed Phase 1 study with 2-h IV infusions (Days 1, 8, 15, q28d, NCT01397929 , CDI-CS-001, Lopez et al. J Clin Oncol 34, 2016 suppl; 2525), dose-limiting vascular effects were observed and appeared Cmax related. Preclinical data suggest that antiproliferative effects of BAL101553/27862 are driven by exposure (AUC); thus vascular toxicity and antitumor activity are mediated by different PK drivers. In this ongoing study (NCT02490800, CDI-CS-002), daily oral BAL101553 was initially examined in solid-tumor patients; no vascular toxicities were observed at doses up to the MAD of 30 mg QD. Given this absence, the study was amended to enroll separate cohorts of patients with progressive or recurrent GBM or high-grade glioma. Methods: This is an ongoing multicenter, open-label, Phase 1 dose-escalation study using a 3+3 design to determine the MTD, characterize dose-limiting toxicities and assess the PK, PD and antitumor activities of daily oral administration of BAL101553 in consecutive 28-day cycles at a starting dose of 8 mg QD. Patients with histologically-confirmed GBM or high-grade glioma, with progressive or recurrent disease after prior RT with/without chemotherapy, are eligible for enrollment. This includes patients with histologically-confirmed low-grade glioma with unequivocal evidence by imaging of transformation to high-grade glioma. Adverse events are assessed using CTCAEv4; tumor response by RANO (every 2 cycles). The dose escalation allows for doubling of dose levels depending on observed toxicities. PD assessments include circulating tumor cells. PK profiles are assessed throughout the first two treatment cycles. Clinical trial information: NCT02490800.

Verification of targeted gene expression profiling panel for identifying biomarker signatures for immunotherapy research.

e23208 Background: Immunotherapy has led to an unprecedented and long-lasting response in susceptible populations. Despite the therapeutic potential of the treatment, identifying biomarkers and stratifying populations that are likely to respond has been a challenge. While gene expression profiling has previously been successfully used to stratify individuals, there exist limitations with the prevalent technologies. In particular, full transcriptome gene expression estimates use limited biological material to measure the concentrations of thousands of uninformative genes and often lacks the depth required to accurately measure expression of lowly-expressed genes. These low-expressing genes may be critical to the identification of a signature associated with susceptible population. Methods: To efficiently measure the expression of the genes potentially informative of an immunotherapy response, we developed a high-throughput targeted gene expression solution measured with our RNA Ion Oncomine™ Immune Response Research Assay panel* containing 395 genes. This panel provides information about the expression of genes involved in tumor checkpoint inhibition, as well as markers of T cell signaling pathway, interferon signaling, tumor infiltrating lymphocytes (TIL). Results: We used publicly-available TCGA data to characterize the complexities of estimating unbiased gene expression from a targeted panel and developed a solution using a new normalization procedure that allows for accurate comparisons of samples within cancer types. Furthermore, we verified that lowering the RNA input amount or changing the assay operator does not contribute to a large variation in the gene expression estimates; each only accounts for less than 10% of variance of the average gene when the assay is compared across biological samples. Conclusions: Creating a panel that achieved high reproducibility and accurate expression estimates of key immune response genes allowed us to accurately separate high and low TIL samples within squamous and adenocarcinoma samples, emphasizing the utility of the panel to biomarker immunotherapy research. *For Research Use Only. Not for use in diagnostic procedures.

A phase I study to assess the safety, pharmacokinetics (PK), pharmacodynamics (PD) and antitumor activities of BAL101553, a novel tumor checkpoint controller (TCC), administered as 48-hour infusion in adult patients with advanced solid tumors.

TPS2602 Background: BAL101553 (prodrug of BAL27862), is a novel TCC that promotes tumor cell death by modulating the spindle assembly checkpoint. BAL27862 has shown potent antitumor activity in diverse preclinical tumor models, including models refractory to standard therapies. In a completed Phase 1 study using 2-h IV infusions (Days 1, 8, 15, q28d, NCT01397929 , CDI-CS-001, Lopez et al. JCO 34, 2016 suppl; 2525) dose-limiting vascular effects were observed and appeared Cmax related. The recommended Phase 2 dose for 2-h IV BAL101553 is 30 mg/m2. Vascular toxicity was not observed in an ongoing study with oral BAL101553 (NCT02490800, CDI-CS-002) at daily doses up to 30 mg (QD). Preclinical data suggest that antiproliferative effects of BAL101553/27862 are driven by exposure (AUC); thus vascular toxicity and antitumor activity are mediated by different PK drivers. BAL27862 has a half-life of ~15 h. Based on PK-modeling, extending the infusion from 2 h to 48 h was expected to result in ~4-fold higher AUC at a given Cmaxlevel and thereby improve the therapeutic window. Methods: This is an ongoing multicenter, open-label, Phase 1 dose-escalation study (NCT02895360, CDI-CS-003/SAKK67/15) using a 3+3 design to determine the MTD, characterize dose-limiting toxicities and assess the PK, PD and antitumor activities of 48-h infusions of BAL101553 in consecutive 28-day cycles at a starting dose of 30 mg/m2 administered on Day 1, 8 and 15 (q28d). The dose escalation scheme foresees up to ~ 50% dose increments depending on observed toxicities. During cycle 2, patients receive 7 days oral (QD) BAL101553 (Day 15–21) instead of the weekly IV infusion to assess absolute oral bioavailability. Patients with histologically-confirmed advanced or recurrent solid tumors are eligible for enrollment. Adverse events are assessed using CTCAEv4; tumor response by RECIST 1.1 (every 2 cycles). PD assessments include optional tumor biopsies and circulating tumor cells. PK profiles are assessed during the first 2 cycles. Two dose cohorts (30 and 45 mg/m2) have completed without DLTs or signs of vascular toxicity. Clinical trial information: NCT02895360.

Phase 1/2a trial of daily oral BAL101553, a novel tumor checkpoint controller (TCC), in advanced solid tumors.

2532 Background: BAL101553 (prodrug of BAL27862) is a small molecule TCC that binds microtubules and promotes tumor cell death by activation of the spindle assembly checkpoint. In a previous study (NCT01397929, Lopez et al. JCO 34, 2016; abstr 2525), 2-h IV infusion on Days 1, 8, 15 (q28d) of BAL101553 up to 80 mg/m2 (maximum administered dose, MAD) showed vascular toxicities, including transient hypertension, which appeared to be Cmax related. The recommended Phase 2 dose (RP2D) was 30 mg/m2 weekly IV. Based on nonclinical models, antiproliferative effects of BAL27862 are driven by AUC. This trial explores whether once daily oral administration of BAL101553 reduces Cmax-related toxicity and improves the therapeutic window (NCT02490800). Methods: Patients (pts) with advanced solid tumors who failed standard therapy, received QD oral BAL101553 (28-d cycles) in a 3+3 dose-escalation design to determine the MTD. Adverse events were assessed by CTCAEv4 grade (G); tumor response by RECIST 1.1; serial PK on Day 1 of Cycles 1 and 2. Results: In the ongoing study, 19 pts (9M/10F; median age 67 y) received doses of 2, 4, 8, 16 or 30 mg oral BAL101553 QD. The MAD was 30 mg with DLTs of reversible G2 hallucination and asymptomatic, reversible G3 electrolyte imbalances. No DLTs were observed at ≤ 16 mg. Dosing is ongoing between 16 and 30 mg QD to determine the MTD. BAL27862 exposures after oral QD dosing of BAL101553 compared to weekly 2-h infusions suggested high relative oral bioavailability. The BAL27862 weekly AUC at the oral MAD (30 mg QD) compared to the RP2D of 30 mg/m2 for 2-h IV was more than 5-fold higher (19,656 vs 3,584 ng*h/mL) and Cmax was 1.5-fold lower (171 vs 266 ng/mL). Both Cmaxand AUC were dose-proportional, with low/moderate variability. Oral BAL101553 had no effects on blood pressure and showed no vascular toxicity. 5 pts had stable disease (2 pts [cholangiocarcinoma, neuroendocrine pancreatic cancer] > 4 cycles). Conclusions: Daily oral BAL101553 enables higher weekly exposures of BAL27862 with lower Cmax levels compared with a 2-h weekly infusion, due to the absence of Cmax related vascular toxicity. Doses up to 16 mg QD are well tolerated. The MAD has been identified as 30 mg QD; definition of the MTD is ongoing. Clinical trial information: NCT02490800.

Abstract A37: CBP501 induces tumor immunogenic cell death and CD8 T cell infiltration into tumor in combination with platinum, thereby increasing the efficacy of immune checkpoint inhibitors against tumors in mice

Abstract Introduction: CBP501, a calmodulin-binding peptide, is an anti-cancer drug candidate that has completed two Phase II clinical trials for patients with malignant pleural mesothelioma and non-small cell lung carcinoma (NSCLC). CBP501 enhances the uptake of platinum agents by cancer cells specifically and induces immunogenic cell death within the clinically achievable dose levels of both CBP501 and cisplatin (CDDP) (EORTC 2015 abstract#C106). Here we treated tumor bearing mice with the combination of CBP501, platinum agents (CDDP and carboplatin), and immune checkpoint inhibitors and analyzed the tumor growth and behaviors of immune cells. Methods: Immuno-competent BALB/c mice were subcutaneously inoculated with CT26WT (5 x105 cells/mouse). A week later, mice were apportioned into 8 groups (6 mice/group) and treated with 2 dosing cycles for 3 anti-cancer agents, alone or in different combinations [CDDP: 4 mg/kg x 1/week, CBP501: 6.0 mg/kg x 1/week, anti-mPD1 mAb (RMP1-14) or anti-PD-L1 mAb (10F.9G2): 200 ug/mice x 2/week]. Results: In BALB/c mice bearing CT26WT tumor, CDDP- or immune checkpoint inhibitor-treated mice showed a minimal anti-tumor effect compared to the vehicle-treated mice. CDDP/immune checkpoint inhibitor-treated mice showed an additive anti-tumor effect. CBP501 significantly enhanced the anti-tumor effect of CDDP/immune checkpoint inhibitor combination. Flow cytometry analysis of single suspension cells from the tumor tissue showed that treatment of CDDP/CBP501 or CDDP/CBP501/anti-mPD1 mAb but not CDDP or anti-mPD1 mAb increased the percentage of tumor-infiltrating CD8+ T cell. Conclusion: An increase of tumor-infiltrating CD8+ T cells in CDDP/CBP501-treated mice supported the conclusion that CDDP/CBP501 treatment induced immunogenic cell death not only in vitro but also in vivo. It also supported the notion that CBP501 would not increase cytotoxicity of platinum agents to non-tumor cells. The fact that CBP501 enhanced anti-tumor activity of immune checkpoint inhibitors in combination with platinum agents (CDDP and carboplatin) would support further clinical development of CBP501. Citation Format: Keiichi Sakakibara, Takuji Sato, Donald W. Kufe, Daniel D. VonHoff, Takumi Kawabe. CBP501 induces tumor immunogenic cell death and CD8 T cell infiltration into tumor in combination with platinum, thereby increasing the efficacy of immune checkpoint inhibitors against tumors in mice. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr A37.

Abstract B17: Validation of targeted gene expression profiling panel for identifying biomarker signatures of immunotherapy responders

Abstract Cancer immunotherapy has led to an unprecedented, long-lasting response in populations susceptible to the therapies. Despite the therapeutic potential, identifying biomarkers and stratifying populations that are likely to respond has been a challenge. Gene expression profiling has previously been successfully used to stratify individuals based on survival and treatment characteristics, but there exist limitations with the prevalent technologies. In particular, full transcriptome gene expression estimates use limited biological material to measure the concentrations of tens of thousands uninformative genes and often lack the depth required to accurately measure expression levels of lowly-expressed genes. These genes may be critical to the identification of a signature associated with immunotherapy responders. To efficiently measure the expression of the key genes potentially informative of an immunotherapy response, we developed a high-throughput targeted gene expression solution measured by our RNA Ion Oncomine Immune Response Profiling panel* containing 395 genes. This panel provides information about the expression of genes involved in tumor checkpoint inhibition (including CTLA4, PD-1, PD-L1, OX-40, 4-1BB, TIM3, LAG3) and other targets such as CSF1R, and IDO1, as well as additional markers of T cell signaling pathway, interferon signaling, and markers of tumor infiltrating lymphocytes (TIL). We used publicly available TCGA data to demonstrate the need and develop a solution for a new normalization procedure that allows for accurate comparisons of samples within various cancer types. Furthermore, we verified a linear and unbiased estimate of fold change in our assays across mixing concentrations of a cell-line titration experiment. Finally, by achieving a high correlation (r > .99) of technical replicates, along with robust expression estimation even at low input amounts (10 ng RNA), our panel offers a valuable solution for biomarker research in cancer immunotherapy. *For research use only. Not for use in diagnostic procedures. Citation Format: Aleksandr Pankov, Yongming Sun, Yuan-Chieh Ku, Warren Tom, Jianping Zheng, Timothy Looney, Janice Au-Yong, Fiona Hyland, Ann Mongan. Validation of targeted gene expression profiling panel for identifying biomarker signatures of immunotherapy responders. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr B17.

Abstract P2-10-01: Genomic profiling of residual ER+ breast cancers treated with prolonged neoadjuvant letrozole reveals novel alterations in clinically resistant tumors

Abstract Background: Approximately 20% of patients with early ER+ breast cancer (BC) treated with adjuvant antiestrogen therapy eventually relapse with endocrine-resistant metastatic disease. We hypothesized that profiling newly diagnosed ER+ BC that persist following prolonged estradiol deprivation with letrozole would identify genomic alterations associated with endocrine resistance. Methods: We treated 57 postmenopausal women (median 77 years; range 60-86) with ER+/HER2– BC with neoadjuvant letrozole (median 7.5 months; range 3-36) followed by surgery and adjuvant endocrine therapy. Patients were followed with serial ultrasounds and defined as non-responders if they developed recurrent locally or metastatic disease, or had a preoperative endocrine prognostic index (PEPI) ≥4 (composite score of post-treatment ER, Ki67, T and N status). Post-treatment specimens were profiled by RNA-seq and targeted capture NGS of >300 cancer-related genes. We screened for variants with a high probability of disrupting protein function (GERP score >4) and excluded likely germline variants by filtering out every alteration not present in COSMIC, if the variant had an allele frequency >0.1% as per the ExAC dataset. Results: Ten patients (17.5%) had a PEPI 0 score, 31 (54%) were PEPI 1-3, and 16 (28%) were PEPI ≥4. After a median follow-up of 50 months (12-100), 9 patients (15.7%) had recurred with metastatic disease (4 with PEPI 1-3, 5 with PEPI ≥4). We identified 294 variants with a median coverage >250x (206 nonsynonymous, 21 nonsense, 58 indels, 8 splice site). Recurrent mutations included PIK3CA (38%), KMT2C (28%), CDH1 (15%), NF1 (12 %), TP53 (10%), MAP3K1 (7%), ERBB2 (7%) and ESR1 (5%). Recurrent amplifications were identified in MCL1 (31%), GNAS (19%), CCND1 (16%), FYN (14%), AURKA (12%), and ERBB2 (10%), while recurrent deletions were found in DUSP4 (12%), NCOR1 (8%) and NF1 (6%). Compared to alterations reported in untreated ER+ breast cancers in TCGA, we observed a significant increase in KMT2C, NF1, MCL1 and FYN alterations (FDR<0.05). MCL1, GNAS and FYN amplifications, DUSP4 deletions, MAP3K1 mutations, and ERBB2 and NF1 alterations were enriched in the non-responder group. Differential expression analysis of the RNA-seq data revealed an enrichment of E2F and MYC target genes, and genes involved in the G2/M checkpoint, TORC1 signaling, EMT and immunosuppression in non-responding tumors. PEPI 0 tumors were enriched with Luminal A subtype tumors, whereas PEPI 4 tumors were enriched with Luminal B, basal and HER2-enriched subtypes. Luminal A tumors exhibited improved disease free survival compared to other subtypes (HR 0.28, 95% CI 0.10-0.64). Gains in the proximal portion of chromosome 1q were associated with poor long-term outcomes as the relapse-free survival rate at 40 months for patients with 1q gains was 89% versus 41% for patients with no 1q gain (p=0.001). Conclusions: Genomic profiling of residual ER+ breast cancers treated with prolonged neoadjuvant letrozole revealed a different mutational landscape than primary untreated ER+ BC. These alterations may be associated with poor response to estrogen deprivation in early breast cancer and deserve further study. Citation Format: Guerrero AL, Stricker T, Hutchinson KE, Formisano L, Giltnane J, Fidalgo A, Schwarz LJ, Gavila J, Guillen V, Lluch A, Ruiz A, Arteaga CL. Genomic profiling of residual ER+ breast cancers treated with prolonged neoadjuvant letrozole reveals novel alterations in clinically resistant tumors [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 P2-10-01.

Abstract 4781: The novel tubulin-binding ‘tumor checkpoint controller’ BAL101553 has anti-cancer activity alone and in combination treatments across a panel of GBM patient-derived xenografts

Abstract Microtubule-targeting agents (MTA) have been employed in the treatment of many cancers for decades. BAL101553 is a highly soluble prodrug of BAL27862, a novel, small molecule, microtubule-depolymerizing agent that induces tumor cell death by activating the ‘spindle assembly checkpoint’. Given intravenously or orally, the drug penetrates the brain and has anti-cancer activity in diverse tumor models refractory to standard MTA or radiotherapy (RT). In this study, BAL101553 was evaluated in orthotopic xenografts from 16 GBM PDX models; 7 of 16 lines demonstrated significant (p<0.01) increases in median survival with BAL101553 versus placebo (range in median survival extension 24-87%). The combination of BAL101553 with conventional therapies for GBM (RT and temozolomide (TMZ) was then evaluated in select lines. In the MGMT methylated GBM12 line, combination of RT with TMZ increased survival compared to placebo (median survival 80 days vs. 23 days, respectively; p<0.001). Extended BAL101553 monotherapy provided a short but significant extension in survival (median survival 31 days, p<0.001), while extended BAL101553 dosing during and after RT/TMZ (median survival 85 days) did not extend survival relative to RT/TMZ alone (p = 0.56). In contrast, in the MGMT unmethylated GBM6 line, combination of RT and extended BAL101553 increased survival (median 90 days, p<0.001) relative to either treatment alone (median survival BAL101553 63 days; RT 69 days) or placebo (46 days). Additionally, the combination of BAL101553 with TMZ (median survival 70 days) was more effective than TMZ alone (median survival 60 days; p = 0.009). Consistent with the unmethylated MGMT status, the TMZ/RT combination (median survival 66 days) was similar to RT alone (p = 0.62), but the combination of extended BAL101553 with RT/TMZ (median survival 101 days; p<0.001 compared to other combination groups) was significantly more effective. To further evaluate whether BAL101553 is a true radiosensitizer, a second GBM6 study was performed. Also here, combination of RT (20Gy, 2wks) with extended BAL101553 dosing (median survival 66 days) significantly extended survival compared to RT alone (median survival 54 days; p = 002). Interestingly, when BAL101553 dosing was limited to 2 weeks with RT, there was no increase in median survival (58 days; p = 0.16). To evaluate effects on tumor repopulation during RT, the efficacy of an extended RT schedule (36 Gy, 6 wks) with or without 6 weeks of BAL101553 was evaluated. In this case, BAL101553 given during the RT schedule (median survival 78 days) extended median survival as compared to RT alone (61 days; p<0.001). Collectively, these data demonstrate that BAL101553 has broad single agent activity across a panel of GBM PDX models and suggests that combination with RT/TMZ therapy may provide additional benefits for survival extension. Citation Format: Ann C. Mladek, Jenny L. Pokorny, Heidi Lane, Felix Bachmann, Mark A. Schroeder, Katrina K. Bakken, Brett L. Carlson, Paul A. Decker, Jeanette E. Eckel-Passow, Jann N. Sarkaria. The novel tubulin-binding ‘tumor checkpoint controller’ BAL101553 has anti-cancer activity alone and in combination treatments across a panel of GBM patient-derived xenografts. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4781.

Vitamin C uncouples the Warburg metabolic switch in KRAS mutant colon cancer.

KRAS mutation is often present in many hard-to-treat tumors such as colon and pancreatic cancer and it is tightly linked to serious alterations in the normal cell metabolism and clinical resistance to chemotherapy.In 1931, the winner of the Nobel Prize in Medicine, Otto Warburg, stated that cancer was primarily caused by altered metabolism interfering with energy processing in the normal cell. Increased cell glycolytic rates even in the presence of oxygen is fully recognized as a hallmark in cancer and known as the Warburg effect.In the late 1970′s, Linus Pauling and Ewan Cameron reported that vitamin C may have positive effects in cancer treatment, although deep mechanistic knowledge about this activity is still scarce.We describe a novel antitumoral mechanism of vitamin C in KRAS mutant colorectal cancer that involves the Warburg metabolic disruption through downregulation of key metabolic checkpoints in KRAS mutant cancer cells and tumors without killing human immortalized colonocytes.Vitamin C induces RAS detachment from the cell membrane inhibiting ERK 1/2 and PKM2 phosphorylation. As a consequence of this activity, strong downregulation of the glucose transporter (GLUT-1) and pyruvate kinase M2 (PKM2)-PTB dependent protein expression are observed causing a major blockage of the Warburg effect and therefore energetic stress.We propose a combination of conventional chemotherapy with metabolic strategies, including vitamin C and/or other molecules targeting pivotal key players involved in the Warburg effect which may constitute a new horizon in anti-cancer therapies.

Phase 1/2a trial of intravenous BAL101553, a novel tumor checkpoint controller (TCC), in advanced solid tumors.

2525Background: BAL101553 (prodrug of BAL27862) is a small molecule TCC that binds microtubules and promotes tumor cell death through activation of the mitotic checkpoint. Antiproliferative effects...

A Phase 1 study to assess the safety, pharmacokinetics (PK), pharmacodynamics (PD) and antitumor activities of daily oral BAL101553, a novel tumor checkpoint controller (TCC) in adult patients with advanced solid tumors.

TPS2594Background: BAL101553 is the pro-drug of BAL27862, a novel small molecule TCC that promotes tumor cell death by modulating the spindle assembly checkpoint (SAC). In a completed Phase 1 (P1) ...

Abstract B159: Modulation of checkpoint expression in tumor microenvironment by intratumoral administration of a novel TLR9 agonist: Rationale for combination therapy

Abstract Recent advances in the understanding of immune tolerance has led to the targeting of various checkpoints for the treatment of cancer. Several independent clinical studies have pointed out a strong correlation between the clinical activity of checkpoint inhibitors and tumor checkpoint expression. These findings highlight an opportunity to improve cancer immunotherapy by combining checkpoint inhibitors with an immunostimulatory agent that modulates the tumor microenvironment. Our objective was to modulate the tumor microenvironment by activating Toll-like receptor (TLR) 9, a pathogen-associated molecular pattern recognition receptor. In preclinical studies, administration of IMO-2125, a novel TLR9 agonist, led to maturation of dendritic cells and induction of IFN-alpha. We hypothesized that immune activation by a TLR9 agonist administered intratumorally (i.t.) will lead to modulation of immune checkpoints in the tumor microenvironment, allowing these tumors to be targeted with a checkpoint inhibitor. To evaluate the potential of i.t. IMO-2125 therapy and its effect on checkpoint gene expression, we employed three different tumor models: CT26 colon carcinoma, A20 lymphoma, and B16 melanoma mouse models. IMO-2125 was administered at a dose of 50 mcg per treatment, either as a single dose or as multiple doses into a targeted tumor nodule. Blood samples were collected post-dosing for analysis of the cytokine profile. Anti-tumor activity was evaluated by measuring the growth of the injected tumor as well as the distant tumor. Tumor nodules were homogenized to analyze gene expression of selected checkpoints by qPCR. Results showed that a single i.t. injection of IMO-2125 in a CT26 colon carcinoma model induced systemic Th1 type cytokines, and resulted in tumor growth inhibition. Two days post IMO-2125 administration, analysis of gene expression of the treated tumor showed upregulation of IDO1 (13 fold) and PDL1 (2.7 fold), and no changes in expression of PD1 and OX40. Increased IDO1 gene expression correlated with increased IDO protein levels in the treated tumor nodules. In contrast, subcutaneous administration of IMO-2125 showed no substantial anti-tumor effect and no significant changes in checkpoint gene expression despite an induction of similar levels of systemic cytokines as the i.t. treatment. Multiple i.t. injections of IMO-2125 led to inhibition of tumor growth of the injected tumor and the distant tumor in all three tumor models. Tumor growth was correlated with increased CD3+ cells in treated as well as distant tumors. Analysis of the checkpoint gene expression at treated and distant tumor sites was conducted at 1-2 weeks post treatment. There was a significant upregulation of IDO1 and PDL1 gene expression in the treated tumors as well as in distant tumors. In addition to IDO1 and PDL1, depending on the tumor model, other immune checkpoints, including PD1, TIM3, BTLA, LAG3, OX40, and CTLA4, were also modulated. In conclusion, IMO-2125 i.t. treatment led to modulation of the tumor microenvironment by inducing local and systemic Th1 type immune responses, which impacted the levels of checkpoint gene expression. Increased expression of some of these checkpoints may allow tumors to be targeted broadly by checkpoint inhibitor therapy. Citation Format: Wayne Jiang, Daqing Wang, Fugang Zhu, Lakshmi Bhagat, Jillian DiMuzio, Sudhir Agrawal. Modulation of checkpoint expression in tumor microenvironment by intratumoral administration of a novel TLR9 agonist: Rationale for combination therapy. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B159.

Abstract A183: The novel tubulin-binding ‘tumor checkpoint controller’ BAL101553 exerts EB1 expression-dependent antitumor effects on glioblastoma stem-like cells in vitro and in vivo

Abstract Background: BAL101553 is the prodrug of the novel small molecule BAL27862, undergoing clinical evaluation in advanced cancer patients as an i.v. (phase 2a) and oral (phase 1) formulation. BAL27862 binds the colchicine site of tubulin with distinct effects on microtubule organization, resulting in activation of the ‘spindle assembly checkpoint’ and tumor cell death. BAL27862 has broad activity against diverse cancer models, including tumors refractory to conventional treatments. The drug efficiently distributes to tumor and to brain, with cytotoxic effects in glioblastoma (GBM) lines. We have shown that End-binding 1-protein (EB1) overexpression correlates with GBM progression and sensitizes GBM tumors to Vinca alkaloids. Moreover, EB1 is overexpressed in the GBM stem-like cells, GBM6, that display a high tumorigenicity with an infiltrative pattern of migration in vivo. Here, we investigate the activity of BAL27862/BAL101553 on GBM6 stem-like cells according to EB1 expression level in vitro and in orthotopically transplanted nude mice. Material and methods: Effects of BAL27862 on wild-type and EB1-downregulated GBM6 cells were analyzed using sulforhodamin B survival, clonogenic and transwell migration assays. Nude mice were orthotopically grafted with GBM6GFPSh0 (EB1+) and GBM6GFPShEB1 (EB1-) cells. BAL101553 (25mg/kg) or vehicle were administrated i.v. at D30, D33 and D36. Stem cell phenotype characterization, tumor volume and brain invasion were analyzed at D45, D75 and D105. Overall survivals were analyzed. Stem-like cell differentiation was analyzed by flow cytometry and real-time PCR by analyzing stem cell markers (A2B5, CD133), markers of differentiation (GFAP, β-III-tubulin and CNPase) and self-renewal assay. Results: BAL27862 inhibited GBM6 survival after 72h (half maximal effective concentration [EC50] = 20nM) with EB1-downregulation resulting in a ∼2-fold increase in EC50 (40nM). BAL27862 inhibited clonogenicity and cell migration in GBM6 cells, even at a low, non-cytotoxic concentration (6 nM). However, these effects were only detectable with cytotoxic concentrations (≥ 20 nM) in EB1 downregulated GBM6 cells. Just three BAL101553 administrations over a week provoked a mice survival increase of 69 and 32 days (vs. vehicle controls) after GBM6GFPSh0 and GBM6GFPShEB1 tumor grafting, respectively. Analysis of the pattern of invasion, and quantification of fluorescent tumor cells and A2B5+cells in brain, demonstrated that control cells were more invasive than EB1-downregulated cells, and that anti-invasive effects of BAL101553 were more potent in control cells than in EB1-downregulated cells. BAL27862 also acted on stem-like cells by inducing apoptosis and differentiation, as shown by the decrease in self renewal and stem cell markers. Conclusion: BAL27862 is the first tubulin-binding agent to show a strong inhibitory activity against stem-like cell proliferation and invasion. These results support further investigation of BAL101553 for the treatment of GBM patients, with EB1 expression a potential predictive biomarker for drug responders. Financial supports: (SIRIC label) INCa-DGOS-Inserm 6038, A*MIDEX project “Investissements d'Avenir” (No. ANR-11-IDEX-0001-02), the ITMO Cancer AVIESAN as part of the Cancer Plan and Basilea Pharmaceutica. Citation Format: Raphael Berges, Aurélie Tchoghandjian, Stephane Honore, Dominique Figarella-Branger, Felix Bachmann, Heidi Lane, Diane Braguer. The novel tubulin-binding ‘tumor checkpoint controller’ BAL101553 exerts EB1 expression-dependent antitumor effects on glioblastoma stem-like cells in vitro and in vivo. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A183.

LY2603618, a selective CHK1 inhibitor, enhances the anti-tumor effect of gemcitabine in xenograft tumor models.

Pharmacological inhibition of CHK1 in the absence of p53 functionality leads to abrogation of the S and G2/M DNA damage checkpoints. We report the preclinical therapeutic activity of LY2603618 (CHK1 inhibitor) at inhibiting CHK1 activation by gemcitabine and enhancing in vivo efficacy. The in vivo biochemical effects of CHK1 inhibition in the absence or presence of DNA damage were measured in human tumor xenograft models. Colon, lung and pancreatic xenografts models were treated with gemcitabine, LY2603618, or gemcitabine plus LY2603618. Gemcitabine treatment alone induced a significant increase in CHK1 autophosphorylation over untreated tumors. Co-administration of LY2603618 with gemcitabine showed a clear inhibition of CHK1 autophosphorylation for at least 24h. Combining LY2603618 with gemcitabine resulted in an increase in H2AX serine 139 phosphorylation, indicating a corresponding increase in damaged DNA in the tumors. LY2603618 abrogated the S-phase DNA damage checkpoint in Calu-6 xenograft tumors treated with gemcitabine but did not significantly alter the G2/M checkpoint. Combining gemcitabine with LY2603618 resulted in a significant increase in tumor growth inhibition in Calu-6, HT-29 and PAXF 1869 xenografts over gemcitabine treatment alone. The best combination efficacy occurred when LY2603618 was given 24h following dosing with gemcitabine. LY2603618 worked effectively to remove the S-phase DNA damage checkpoint and increase the DNA damage and the antitumor activity of gemcitabine treatment.

Distribution and prognostic relevance of tumor-infiltrating lymphocytes (TILs) and PD-1/PD-L1 immune checkpoints in human brain metastases.

The activation of immune cells by targeting checkpoint inhibitors showed promising results with increased patient survival in distinct primary cancers. Since only limited data exist for human brain metastases, we aimed at characterizing tumor infiltrating lymphocytes (TILs) and expression of immune checkpoints in the respective tumors. Two brain metastases cohorts, a mixed entity cohort (n = 252) and a breast carcinoma validation cohort (n = 96) were analyzed for CD3+, CD8+, FOXP3+, PD-1+ lymphocytes and PD-L1+ tumor cells by immunohistochemistry. Analyses for association with clinico-epidemiological and neuroradiological parameters such as patient survival or tumor size were performed. TILs infiltrated brain metastases in three different patterns (stromal, peritumoral, diffuse). While carcinomas often show a strong stromal infiltration, TILs in melanomas often diffusely infiltrate the tumors. Highest levels of CD3+ and CD8+ lymphocytes were seen in renal cell carcinomas (RCC) and strongest PD-1 levels on RCCs and melanomas. High amounts of TILs, high ratios of PD-1+/CD8+ cells and high levels of PD-L1 were negatively correlated with brain metastases size, indicating that in smaller brain metastases CD8+ immune response might get blocked. PD-L1 expression strongly correlated with TILs and FOXP3 expression. No significant association of patient survival with TILs was observed, while high levels of PD-L1 showed a strong trend towards better survival in melanoma brain metastases (Log-Rank p = 0.0537). In summary, melanomas and RCCs seem to be the most immunogenic entities. Differences in immunotherapeutic response between tumor entities regarding brain metastases might be attributable to this finding and need further investigation in larger patient cohorts.

Abstract 3789: BAL101553 (prodrug of BAL27862): the spindle assembly checkpoint is required for anticancer activity

Abstract Background: BAL101553 is a highly soluble prodrug of BAL27862, a novel, small molecule, microtubule (MT)-depolymerizing agent with a broad in vitro anti-proliferative activity against human tumor lines refractory to standard MT-targeting agents. The prodrug has anti-cancer activity in diverse tumor models when given orally or iv; alone or in combination therapy. In this study, the role of the ‘Spindle Assembly Checkpoint’ (SAC) in the mechanism of action of BAL27862 was evaluated. Methods: To evaluate BAL27862-resistance mechanisms, tumor lines were selected in vitro by increasing drug levels. Anti-proliferative activity was analyzed using Crystal Violet assay. siRNA transfection for 24 h was followed by drug treatment (50 nM) for 48 h. Mitotic index was evaluated after 18 h treatment by p-Histone H3 staining (pHH) or flow cytometry (FC). SAC complex formation was assessed by BubR1 immunoprecipitation (IP) and immunoblotting for SAC components. Results: The SAC ensures cell cycle arrest upon spindle disruption, promoting tumor cell death after prolonged mitotic arrest. SAC formation involves phosphorylation and assembly of a multiprotein complex, of which BubR1 and Mad2 are essential components. Using siRNA approaches, BubR1 was shown to be required for BAL27862 anti-proliferative activity in 5 BAL27862 sensitive tumor lines (IC50s: 8-20 nM). A similar analysis of Mad2 in 3 lines resulted in almost identical attenuation of the effect of BAL27862. For example, non-targeting control siRNA (NTC)-treated H460 lung cancer cells entered a cell death program following BAL27862 treatment (∼45% cell death vs. starting cell number); a response attenuated by BubR1 and Mad2 down regulation (71% and 62% proliferation resp. vs. DMSO controls). The accumulation of mitotic arrested (pHH positive) cells associated with BAL27862 treatment was also absent after BubR1 or Mad2 siRNA transfection. To investigate whether BAL27862 (50 nM, 18 h) induces the formation of the SAC, BubR1 IPs from 3 sensitive tumor lines (IC50: HeLa cervical: 20 nM; A549 lung: 15 nM; SKOV3 ovarian: 12 nM) were analyzed for SAC components. In all cases, complex formation was observed, consistent with a concomitant mitotic accumulation as measured by FC. Strikingly, in variants of the same lines selected for BAL27862 resistance (resistance factors: 3.2-9.4) no SAC formation or mitotic accumulation was observed under the same conditions. Moreover, BAL27862-induced protein mobility shifts, consistent with activating phosphorylation of SAC components, were also absent in the resistant variants. Conclusions: The association of SAC activation with BAL101553sensitivity suggests prediction of cancer response may lie in its tumor checkpoint control function. BAL101553 is currently in Phase 2a clinical evaluation in advanced cancer patients. Exploratory biomarkers are being evaluated to aid selection of patients most likely to respond. Citation Format: Felix Bachmann, Karin Burger, Heidi Lane. BAL101553 (prodrug of BAL27862): the spindle assembly checkpoint is required for anticancer activity. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3789. doi:10.1158/1538-7445.AM2015-3789

Abstract 1674: Humanized mouse models for personalized preclinical testing of monoclonal antibodies targeting immune checkpoints

Abstract The blockade of immune checkpoints with monoclonal antibodies (mAbs) is a promising therapeutic avenue, with durable objective responses observed in patients with solid tumors, particularly melanoma, non-small cell lung cancer (NSCLC) and renal cell carcinoma. Preclinical models that recapitulate a functional human immune system will therefore be essential tools for the continued investigation of immunotherapy approaches. Champions Oncology is engaged in advanced personalized solutions and our TumorGraft models have been developed and extensively characterized as a platform for use in personalizing cancer patient care, as well as pharmaceutical development. However, because TumorGrafts are established by engrafting patient tumors into immune-deficient mice, the therapeutic efficacy of immune-modulatory drugs cannot be directly examined. To circumvent this limitation, we reconstituted the human immune system by engrafting human hematopoietic cells (HLA-A2; CD34+) into immune-compromised mice (PrkdcscidIl2rgtm1Sug) carrying the scid mutation and a targeted mutation of the Il2r-gamma gene. Ten to twelve weeks later, mature CD45+ human T cells could be detected in these mice, at which time TumorGrafts were established, followed by drug-sensitivity testing with various mAbs targeting the immune system. Fifty six well-established melanoma, colorectal, breast and lung TumorGraft models were characterized and selected with regard to their HLA type and other molecular characteristics such as BRAF mutation status (in melanoma) and KRAS mutation status (in colorectal and lung cancer) as well as expression of PD-L1. With the present study, we demonstrated the potential of combining the humanized mouse with Champions TumorGraft to generate a preclinical platform for assessing the therapeutic value of mAbs targeting immune checkpoints in various solid tumors. Citation Format: Gilson S. Baia, David Vasquez, Daniel Ciznadija, Brandy Wilkinson, David Sidransky, Amanda Katz, Keren Paz. Humanized mouse models for personalized preclinical testing of monoclonal antibodies targeting immune checkpoints. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1674. doi:10.1158/1538-7445.AM2014-1674

Genome-Wide shRNA Screen Identifies WEE1 As a Critical Mediator of Cell Fate and Novel Therapeutic Target in AML,

Abstract 3503Acute myeloid leukemia (AML) remains a therapeutic challenge despite increasing knowledge of the molecular origins of the disease, as the mechanisms of AML cell escape from chemotherapy remain poorly defined. We hypothesized that AML cells are addicted to specific molecular pathways in the context of chemotherapy and used complementary approaches to identify these addictions. Using novel molecular and computational strategies, we performed genome-wide shRNA screens to identify proteins which mediate AML cell fate in cytarabine in 2 AML cell lines (Molm13 and MV4-11). Over 55,000 shRNAs targeting over 11,000 genes were quantified by deep sequencing to identify shRNAs under-represented in the context of cytarabine as compared to no treatment. Complementary analyses identified 125 genes as mediators of AML cell fate in cytarabine. In addition we performed gene expression profiling of AML cells exposed to cytarabine to identify genes with induced expression in this context and examined existing gene expression data from primary patient samples. The integration of these independent analyses strongly implicates cell cycle checkpoint proteins, particularly WEE1, as critical mediators of AML cell fate in cytarabine. Knockdown of WEE1 in a secondary screen confirmed its role in AML cell survival in cytarabine. Pharmacologic inhibition of WEE1 in several, but not all, AML cell lines is synergistic with cytarabine, suggesting underlying molecular susceptibility to this combination of drugs. A WEE1 inhibitor is in Phase II trials in solid tumors, primarily as a means to abrogate the G2/M checkpoint in tumors with TP53 dysfunction. Further experiments demonstrate that inhibition of WEE1 prevents slowed S-phase progression induced by cytarabine in AML cells, broadening the functions of WEE1 that may be exploited therapeutically. Preliminary experiments indicate synergistic inhibition of AML cellular proliferation with daunorubicin in some AML cell lines. Experiments to determine whether WEE1 inhibition in combination with chemotherapy prolongs survival of mice with leukemia are underway. These data highlight the power of integrating functional and descriptive genomics, and identify WEE1 as potential therapeutic target in AML. Disclosures:No relevant conflicts of interest to declare.

TLX1-Induced T-cell Acute Lymphoblastic Leukemia

The TLX1 transcription factor oncogene is frequently activated by chromosomal translocations in T-cell acute lymphoblastic leukemia (T-ALL) and defines a distinct molecular group of tumors characterized by differentiation arrest at the early cortical stage of thymocyte differentiation and excellent response to therapy. Recent developments from the analysis of genomic data on TLX1-specific transcriptional targets and analysis of the molecular mechanisms of TLX1 transformation in human- and mouse-induced leukemias have shown novel insight into the activity of this transcription factor oncogene. Aberrant expression of TLX1 in T-cell progenitors disrupts normal T-cell development and triggers the development of aneuploidy during T-cell transformation. Importantly, the disruption of the mitotic checkpoint in TLX1-induced tumors may be linked not only to the acquisition of secondary genetic alterations in T-ALL but also to increased sensitivity of these tumors to chemotherapy with drugs targeting the formation of the mitotic spindle.