Lata Jayaraman

Dissecting Therapeutic Resistance to ERK Inhibition.

The MAPK pathway is frequently activated in many human cancers, particularly melanomas. A single-nucleotide mutation in BRAF resulting in the substitution of glutamic acid for valine (V600E) causes constitutive activation of the downstream MAPK pathway. Selective BRAF and MEK inhibitor therapies have demonstrated remarkable antitumor responses in BRAFV600E-mutant melanoma patients. However, initial tumor shrinkage is transient and the vast majority of patients develop resistance. We previously reported that SCH772984, an ERK 1/2 inhibitor, effectively suppressed MAPK pathway signaling and cell proliferation in BRAF, MEK, and concurrent BRAF/MEK inhibitor-resistant tumor models. ERK inhibitors are currently being evaluated in clinical trials and, in anticipation of the likelihood of clinical resistance, we sought to prospectively model acquired resistance to SCH772984. Our data show that long-term exposure of cells to SCH772984 leads to acquired resistance, attributable to a mutation of glycine to aspartic acid (G186D) in the DFG motif of ERK1. Structural and biophysical studies demonstrated specific defects in SCH772984 binding to mutant ERK. Taken together, these studies describe the interaction of SCH772984 with ERK and identify a novel mechanism of ERK inhibitor resistance through mutation of a single residue within the DFG motif. Mol Cancer Ther; 15(4); 548–59. ©2016 AACR.

CRLX101, a Nanoparticle–Drug Conjugate Containing Camptothecin, Improves Rectal Cancer Chemoradiotherapy by Inhibiting DNA Repair and HIF1α

Novel agents are needed to improve chemoradiotherapy for locally advanced rectal cancer. In this study, we assessed the ability of CRLX101, an investigational nanoparticle-drug conjugate containing the payload camptothecin (CPT), to improve therapeutic responses as compared with standard chemotherapy. CRLX101 was evaluated as a radiosensitizer in colorectal cancer cell lines and murine xenograft models. CRLX101 was as potent as CPT in vitro in its ability to radiosensitize cancer cells. Evaluations in vivo demonstrated that the addition of CRLX101 to standard chemoradiotherapy significantly increased therapeutic efficacy by inhibiting DNA repair and HIF1α pathway activation in tumor cells. Notably, CRLX101 was more effective than oxaliplatin at enhancing the efficacy of chemoradiotherapy, with CRLX101 and 5-fluorouracil producing the highest therapeutic efficacy. Gastrointestinal toxicity was also significantly lower for CRLX101 compared with CPT when combined with radiotherapy. Our results offer a preclinical proof of concept for CRLX101 as a modality to improve the outcome of neoadjuvant chemoradiotherapy for rectal cancer treatment, in support of ongoing clinical evaluation of this agent (LCC1315 NCT02010567). Cancer Res; 77(1); 112-22. ©2016 AACR.

A Brain Penetrant Mutant IDH1 Inhibitor Provides In Vivo Survival Benefit

Mutations in IDH1 are highly prevalent in human glioma. First line treatment is radiotherapy, which many patients often forego to avoid treatment-associated morbidities. The high prevalence of IDH1 mutations in glioma highlights the need for brain-penetrant IDH1 mutant-selective inhibitors as an alternative therapeutic option. Here, we have explored the utility of such an inhibitor in IDH1 mutant patient-derived models to assess the potential therapeutic benefits associated with intracranial 2-HG inhibition. Treatment of mutant IDH1 cell line models led to a decrease in intracellular 2-HG levels both in vitro and in vivo. Interestingly, inhibition of 2-HG production had no effect on in vitro IDH1 mutant glioma cell proliferation. In contrast, IDH1 mutant-selective inhibitors provided considerable survival benefit in vivo. However, even with near complete inhibition of intratumoral 2-HG production, not all mutant glioma models responded to treatment. The results suggest that disruption of 2-HG production with brain-penetrant inhibitors in IDH1 mutant gliomas may have substantial patient benefit.

Abstract 3209: CRLX101, an investigational nanoparticle-drug conjugate of camptothecin, demonstrates synergy with immunotherapy agents in preclinical models

CRLX101, an investigational nanoparticle-drug conjugate (NDC) containing the payload camptothecin, is currently being clinically evaluated in multiple treatment-refractory solid tumors. CRLX101 has been shown preclinically to be active in many different tumor types as a dual inhibitor of topoisomerase 1 and hypoxia-inducible factor 1α (HIF-1α). It has a long circulation half-life and has been shown pre-clinically to release camptothecin in a slow and prolonged manner in tumors. Camptothecin itself was identified as an active anti-tumor agent preclinically but was not developed clinically due to its poor tolerability in patients. The development of CRLX101, which has not shown significant toxicity in over 300 patients to date, offers a unique opportunity to improve cancer treatment in a meaningful way. Recent publications have suggested that tumor expression of the immune-suppressive molecule PD-L1 is controlled by HIF-1α. Since CRLX101 is a potent inhibitor of HIF-1α, it is possible that CRLX101 behaves as an inhibitor of the PD-1/PD-L1 axis in vivo. We therefore hypothesized that a combination of CRLX101 with agents that are being investigated in combination with the anti PD-1 antibody would lead to increased efficacy. Indole diamine oxygenase (IDO) inhibitors are a new class of drugs that decrease tumor-induced immune suppression and are currently being evaluated in the clinic with anti-PD-1 antibodies. Using syngeneic tumor models, we tested the combination of CRLX101 with three different IDO inhibitors. Treatment of the B16.F10 melanoma model with IDO inhibitors NLG919, INCB024360 or indoximod had no effect on tumor growth while CRLX101 as monotherapy showed moderate anti-tumor activity. When CRLX101 was combined with any of the three IDO inhibitors, the anti-tumor activity was greatly improved compared to monotherapy. Similar results were noted in other syngeneic tumor models. This improved combination response was not observed in IDO inhibitor combinations with the clinically approved topoisomerase inhibitor irinotecan, suggesting that CRLX101 provides a unique advantage in this context. Interestingly, CRLX101/IDO inhibitor combination was also superior to the combination of anti-PD-1 antibody and IDO inhibitor. These data suggest that CRLX101 in combination with IDO inhibitors can successfully block host-mediated immune-suppression to enhance anti-tumor immunity, and this combination may therefore show improved therapeutic activity in the clinic. We therefore plan to explore the immune-specific effects of CRLX101 as well as the mechanistic basis for the observed combination response, and use that information to guide CRLX101 clinical strategy in the setting of chemo-immunotherapy regimens. Citation Format: Douglas Lazarus, Christian Peters, Adam Stockmann, Scott Eliasof, Lata Jayaraman. CRLX101, an investigational nanoparticle-drug conjugate of camptothecin, demonstrates synergy with immunotherapy agents in preclinical models. [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 3209.

Abstract PR06: Drugging the human microbiome for combination with tumor immunotherapy

The human gut microbiome is a diverse, dynamic, and complex ecosystem that modulates host processes including metabolism, inflammation, and cellular and humoral immune responses. Recent studies have suggested that the microbiome may also influence the development of certain cancers such as colorectal cancer, and equally importantly, tumor response to systemic therapy, especially immunotherapy. Multiple groups are exploring the therapeutic utility of the microbiome to enhance clinical response through the use of defined oral therapeutics comprising living commensal bacteria, which would represent a new therapeutic modality. Exploiting the microbiome for therapeutic benefit is not without its challenges due to the heterogeneity of the gut microbiota across healthy donors and patients. In addition, many aspects of conventional small molecule and biologics drug discovery and development do not apply to this novel class of living drugs. We present an approach that leverages the concept of “reverse translation,” using genomic and immunologic characterization of patient samples from interventional studies to define and better understand the organisms and mechanisms that contribute to response or non-response to immunotherapy. We are investigating the relationship between the composition of the gut microbiome prior to therapy and the antitumor response in patients receiving checkpoint inhibitors (CPI), as well as how CPI treatment modulates the microbiome in both responders and nonresponders. Fecal and blood samples are collected before and during therapy from cancer patients who receive approved CPI; tumor types include renal, bladder, and NSCLC. Whole metagenomic shotgun sequencing of patient microbiomes is used to identify higher order (e.g., order- and family-level) “microbial signatures” that associate with response to CPI treatment. We then utilize proprietary algorithms that enable species- and strain-level resolution of microbial signatures. In addition, global and targeted metabolomics are used to identify functional pathways associated with outcome, and these pathways can be linked to species and strains identified by genomic analysis. Our discovery strategy iterates computational analyses and machine learning approaches with empirical in vitro and ex vivo screening of strains and consortia to inform selection and drive drug design. Data from such a comprehensive approach is invaluable for designing compositions of bacteria that form “functional ecological networks” that can impact response to CPI therapy. Finally, our microbial library of >14,000 isolates from healthy human subjects captures the phylogenetic diversity and functional breadth of the gastrointestinal microbiome, and provides a robust platform to build unique compositions. Such compositions, when tested in syngeneic tumor models in germ-free mice, can provide a preliminary readout of the contributions of members of the consortia and enable candidate identification. We present examples of reverse translation in patients with recurrent Clostridium difficile infection and ulcerative colitis, a form of inflammatory bowel disease, that have led to the translation of three drugs that are currently in clinical trials. This roadmap provides insight into how similar drugs can be discovered and developed in the setting of immunotherapy to augment the efficacy of CPIs by altering the cancer-immune set point. This abstract is also being presented as Poster A06. Citation Format: David N. Cook, Jonathan Peled, Marcel van den Brink, Lata Jayaraman. Drugging the human microbiome for combination with tumor immunotherapy [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr PR06.

Abstract PR10: Significant improvements in therapeutic index for conjugated payloads using a nanoparticle-drug conjugate (NDC) platform to provide sustained drug release and potentially improved anticancer effects

Cerulean Pharma Inc.9s Dynamic Tumor Targeting™ Platform creates nanoparticle-drug conjugates (NDCs) designed to significantly mitigate a payload9s limitations by providing sustained drug delivery to the tumor and superior therapeutic index through controlled release kinetics. By conjugating drug payloads to our novel β-cyclodextrin-PEG copolymer through linker strategies that allow modulation of release and pharmacokinetics (PK), we provide advantages over entrapped nanoparticle strategies, e.g., polymeric nanoparticles and liposomes. Cerulean has two NDCs in the clinic, CRLX101 and CRLX301, evidencing the translatability of our technology. CRLX101 has been dosed in over 350 patients and CRLX301 is in an ongoing Phase 1/2a trial. Our Dynamic Tumor Targeting Platform is applicable to a diverse range of drug payload possibilities, including small molecules with a host of physiochemical properties, i.e., hydrophobic and hydrophilic payloads, and functional groups with chemical handles for conjugation. To illustrate the capabilities of our platform we will present the biological impact of a series of cabazitaxel-containing NDCs with linkers encompassing a diversity of in vitro release rates. In vivo PK studies showed high and sustained levels of released drug in tumor tissues (>168 hrs), and in vivo mouse tumor model studies demonstrated vastly improved efficacy, i.e., tumor regression and significant tumor growth delay, and survival over separately dosed cabazitaxel including efficacy in a taxane-resistant tumor model. Cerulean continues to expand its platform through the development of new and emerging capabilities to treat patients living with cancer. In this regard, we also will address the future evolution of NDCs, including the conjugation of multiple payloads to a single NDC and the development of antibody-conjugated NDCs. This abstract is also being presented as Poster B43. Citation Format: Chester Metcalf, III, Derek van der Poll, Liang Zhao, Tiffany Halo, Doug Lazarus, Adam Stockmann, Christian Peters, Donna Brown, Roy Case, Ellen Rohde, Lata Jayaraman, Hongwei Wang, Tiffany Crowell, Adrian Senderowicz, Scott Eliasof. Significant improvements in therapeutic index for conjugated payloads using a nanoparticle-drug conjugate (NDC) platform to provide sustained drug release and potentially improved anticancer effects. [abstract]. In: Proceedings of the AACR Special Conference on Engineering and Physical Sciences in Oncology; 2016 Jun 25-28; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2017;77(2 Suppl):Abstract nr PR10.

Abstract 1345: Tumor selective localization of CRLX101, an investigational nanoparticle-drug conjugate of camptothecin

CRLX101, an investigational nanoparticle-drug conjugate (NDC) containing the payload camptothecin, is currently being clinically evaluated in multiple treatment-refractory solid tumors. In preclinical models, CRLX101 is believed to release camptothecin in the tumor in a slow and prolonged manner due to its long circulation half-life. CRLX101 has been shown preclinically to be a dual inhibitor of topoisomerase 1 and hypoxia-inducible factor 1α. It has demonstrated striking anti-tumor activity in several different tumor models. Camptothecin itself was identified as an active anti-tumor agent preclinically but was not developed clinically due to its poor tolerability in patients. The development of CRLX101, which has not shown significant toxicity in over 300 patients to date, offers a unique opportunity to improve cancer treatment in a meaningful way. We hypothesized that CRLX101 utilizes the enhanced permeability and retention (EPR) effect to accumulate selectively in tumors. In this study, we sought to mechanistically dissect the process of CRLX101 entry and accumulation into tumor cells using multiple methods, both in vitro and in xenograft tumors in vivo. Using confocal microscopy, we detected camptothecin fluorescence in CRLX101-treated tumor cells in culture as well as in tumor tissue from mice treated with CRLX101. We can co-localize this camptothecin with intact nanoparticles using an anti-PEG antibody that specifically detects the PEG loops in the NDCs. More recently, we have shown that camptothecin and anti-PEG co-localize specifically in tumors of patients treated with CRLX101 but not in adjoining normal tissue. We can also demonstrate that macropinocytosis and activation of actin polymerization play a role in the process by which tumor cells take up CRLX101. Using an anti-CD31 antibody, we can visualize the distance traversed by CRLX101 from the tumor vasculature over time. We have developed novel analytical methods to precisely quantify both released and CRLX101-conjugated camptothecin over time in CRLX101 treated tumor cells in vitro, as well as in tumor tissue from mice treated with CRLX101 in vivo. Using cell viability assays, we can correlate the kinetics of camptothecin released inside tumor cells to the degree of tumor cell kill. We believe that these data are an important step forward in understanding the precise mechanism(s) underlying selective delivery of CRLX101 into tumor tissue. Citation Format: Christian G. Peters, Douglas Lazarus, Donna Brown, Ningning Zhang, Adam P. Stockmann, Roy Case, Ellen Rohde, Scott Eliasof, Lata Jayaraman. Tumor selective localization of CRLX101, an investigational nanoparticle-drug conjugate of camptothecin. [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 1345.

Abstract CT090: Phase II trial of the NDC CRLX101 in combination with bevacizumab in patients with platinum-resistant ovarian cancer (PROC)

Background: CRLX101 is an investigational nanoparticle-drug conjugate (NDC) with a camptothecin payload. CRLX101 is a dual inhibitor of topoisomerase 1 and hypoxia inducible factors 1α and 2α (HIF1-α and HIF-2α). CRLX101 is being explored clinically in several tumor types including renal cell carcinoma (RCC) in combination with Avastin®, (Keefe, ASCO 2015, 4543) and platinum-resistant ovarian cancer (PROC) (Krasner ASCO 2014, 5581). The PROC study has a CRLX101 monotherapy arm (Group A) and a combination arm with Avastin (Group B). Group A patients (pts) received CRLX101 at 15mg/m2 every other week and Group B patients received CRLX101 15 mg/m2 with Avastin 10 mg/kg every other week until progression or unacceptable toxicity. The primary endpoint for both groups was rate of progression free survival at 6 months (PFS6) using RECIST 1.1 criteria. Secondary endpoints were objective response rate (ORR), PFS, ?50% reduction of CA125 over baseline, and safety. Adverse events (AEs) were assessed by CTCAE v4.0. Pre- and post-treatment tumor biopsies were collected from a cohort of patients in Group A to evaluate relevant PD endpoints, suggesting that CRLX101 is active in PROC. Results: A total of 18 PROC patients were evaluated in Group B. Demographics: Median age of the patient was 59years (range: 46-68). Median number of previous regimens was 2 (range: 1-3), ECOG PS 0 (13 pts) or 1 (5 pts). The PFS6 was demonstrated in 56% pts (10 out of 18). The PR and stable disease (SD) rates were 17% (3 out of 18 pts) and 78% (14 out of 18 pts), respectively. Median progression-free survival is 6.2 months to date. A ? 50% decline in CA125 was demonstrated in 44% pts (8 out of 18). AEs most commonly observed with the combination were nausea (10 pts, 56%), anemia (10 pts, 56%); proteinuria (8 pts, 44%) and fatigue (6 pts, 33%). The majority of AEs were Grade 1. There were only two drug-related AEs Grade ? 3: one patient developed uncomplicated and reversible elevated ALT (grade 3) and another patient had a short-lived decrease in ANC (grade 4). A retrospective analysis for both cohorts, Group A vs. Group B, demonstrated that the combination showed increased antitumor activity: PFS6: 27 vs 56%, mPFS 4.2 vs 6.2 months and CA125: 23 vs 44%. Conclusions: CRLX101 15 mg/m2 in combination with Avastin 10 mg/kg given every other week demonstrated antitumor activity and was generally well-tolerated in advanced PROC pts. An additional 25 pts will be enrolled in Group B. This combination is currently being explored in an ongoing randomized controlled Phase 2 trial in 3rd and 4th line RCC, and is also being tested in combination with weekly paclitaxel in an ongoing Phase 1b trial in PROC. Clinical trial information: NCT01652079 Citation Format: Carolyn Krasner, Michael Birrer, Christian Peters, Lata Jayaraman, Scott Eliasof, Andres Tellez, William Downing, Adrian Senderowicz. Phase II trial of the NDC CRLX101 in combination with bevacizumab in patients with platinum-resistant ovarian cancer (PROC). [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 CT090.

Abstract 2343: A novel ERK inhibitor is active in models of acquired resistance to BRAF and MEK inhibitors.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC The high frequency of activating BRAFV600E mutations in melanoma (40-70%), thyroid (50%) and colorectal cancer (10%), or KRAS/NRAS mutations in melanoma (20%), pancreatic (90%), colorectal (50%) and non-small cell lung cancer (30%), provides strong rationale for targeting the MAPK pathway as a therapeutic strategy 1-6. Vemurafenib (PLX4032) and dabrafenib (GSK2118436), selective BRAF inhibitors, and trametinib (GSK1120212), an allosteric MEK inhibitor, have shown robust clinical efficacy in melanoma patients 7-10. However, the majority of responses are transient and cellular resistance is often associated with pathway reactivation involving the downstream extracellular-signal-regulated kinases 1 and 2 (ERK1/2) (reviewed in 11). We hypothesized that pathway blockade at ERK, the last signaling node prior to MAPK transcriptional programming, would not only be efficacious in MAPK-activated tumors but would also have utility in BRAF or MEK inhibitor resistant settings. We therefore sought to identify small molecule inhibitors of ERK. This report describes the identification and characterization of SCH772984, a potent and selective ATP competitive inhibitor of ERK1/2 which displays behaviors of both type I and type II kinase inhibitors. SCH772984 has nanomolar cellular potency on tumor cells with mutations in BRAF, NRAS, or KRAS and induces tumor regressions in xenograft models at tolerated doses. Importantly, SCH772984 effectively inhibited MAPK signaling and cell proliferation in BRAF or MEK inhibitor resistant models as well as in the context of BRAF/MEK combination resistance. Together these data support the clinical development of ERK inhibitors, not only in patients with MAPK activated tumors, but also in patients who have developed acquired resistance to BRAF or MEK inhibitors or resistance to the recently described combination of these agents. Citation Format: Ahmed A. Samatar, Erick J. Morris, Sharda Jha, Restaino R. Clifford, Bart Luttrerbach, Marc Pelletier, Ulrike Philippar, Lata Jayaraman, Leigh Zawel, Steve Fawell, Gary Gilliland. A novel ERK inhibitor is active in models of acquired resistance to BRAF and MEK inhibitors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2343. doi:10.1158/1538-7445.AM2013-2343