Noel R. Monks

Potent cytotoxicity of the phosphatase inhibitor microcystin LR and microcystin analogues in OATP1B1- and OATP1B3-expressing HeLa cells

Microcystins are a family of cyclic peptides that are potent inhibitors of the protein phosphatase families PP1 and PP2A. Only three human proteins are thought to be able to mediate the hepatic uptake of microcystins (the organic anion-transporting polypeptides OATP1B1, OATP1B3, and OATP1A2), and the predominant hepatic expression of these transporters accounts for the liver-specific toxicity of microcystins. A significant obstacle in the study of microcystins as anticancer drugs is the requirement of specific transport proteins for cellular uptake. We report that OATP1B3 mRNA is up-regulated in non–small cell lung cancer tumors in comparison with normal control tissues. This finding led to the exploration of microcystins as potential anticancer agents. We have developed a HeLa cell model with functional OATP1B1 and OATP1B3 activity. Transiently transfected HeLa cells are over 1,000-fold more sensitive to microcystin LR than the vector-transfected control cells, showing that transporter expression imparts marked selectivity for microcystin cytotoxicity. In addition, microcystin analogues showed variable cytotoxicities in the OATP1B1- and OATP1B3-transfected cells, including two analogues with IC50 values <1 nmol/L. Cytotoxicity of microcystin analogues seems to correlate to the inhibition of PP2A in these cells and induces rapid cell death as seen by chromatin condensation and cell fragmentation. These studies show that microcystin-induced phosphatase inhibition results in potent cytotoxicity when microcystin compounds can gain intracellular access and are a potent novel class of therapeutic agents for tumors expressing these uptake proteins. [Mol Cancer Ther 2007;6(2):587–98]

Melanoma genotypes and phenotypes get personal

Traditionally, the diagnosis of metastatic melanoma was terminal to most patients. However, the advancements towards understanding the fundamental etiology, pathophysiology, and treatment have raised melanoma to the forefront of contemporary medicine. Indeed, the evidence of durable remissions are being heard ever more frequently in clinics around the globe. Despite having more gene mutations per cell than any other type of cancer, investigators are overcoming complex genomic landscapes, signaling pathways, and immune checkpoints by generating novel technological methods and clinical protocols with breath-taking speed. Significant progress in deciphering molecular genetics, epigenetics, kinase-driven networks, metabolomics, and immune-enhancing pathways to achieve personalized and positive outcomes has truly provided new hope for melanoma patients. However, obstacles requiring breakthroughs include understanding the influence of sunlight exposure on melanoma etiology, and overcoming all too frequently acquired drug resistance, complicating targeted therapy. Pathologists continue to have critically important roles in advancing the field, particularly in the area of transitioning from microscope-based diagnostic reports to pharmacogenomics through molecularly informed tumor boards. Although melanoma is no longer considered just ‘one disease’, pathologists will continue this rapidly progressing and exciting journey to identify tumor subtypes, to utilize tumorgraft or so-called patient-derived xenograft (PDX) models, and to develop companion diagnostics to keep pace with the bewildering breakthroughs occurring on a regular basis. Exactly which combination of drugs will ultimately be required to eradicate melanoma cells remains to be determined. However, it is clear that pathologists who are as dedicated to melanoma as the pioneering pathologist Dr Sidney Farber was committed to childhood cancers, will be required as the battle against melanoma continues. In this review, we describe what sets melanoma apart from other tumors, and demonstrate how lessons learned in the melanoma clinic are being transferred to many other types of aggressive neoplasms.

Characterization of the anti‐CD22 targeted therapy, moxetumomab pasudotox, for B‐cell precursor acute lymphoblastic leukemia

Moxetumomab pasudotox is a second‐generation recombinant immunotoxin against CD22 on B‐cell lineages. Antileukemic activity has been demonstrated in children with chemotherapy‐refractory B‐cell precursor acute lymphoblastic leukemia (BCP‐ALL), with variable responses. Here, we report in vitro and in vivo evaluation of moxetumomab pasudotox treatment of human cell lines and patient‐derived cells as a preliminary study to understand characteristics of sensitivity to treatment. Binding, internalization, and apoptosis were evaluated using fluorescently tagged moxetumomab pasudotox. Studies in NOD‐scid IL2Rgnull mice showed a modest survival benefit in mice engrafted with 697 cells but not in NALM6 or the two patient‐derived xenograft models.

Antitumor Activity of MEDI3726 (ADCT-401), a Pyrrolobenzodiazepine Antibody–Drug Conjugate Targeting PSMA, in Preclinical Models of Prostate Cancer

Prostate-specific membrane antigen (PSMA) is a membrane-bound glutamate carboxypeptidase that is highly expressed in nearly all prostate cancers with the highest expression in metastatic castration-resistant prostate cancer (mCRPC). The prevalence of increased surface expression and constitutive internalization of PSMA make it an attractive target for an antibody–drug conjugate (ADC) approach to treating patients with mCRPC. MEDI3726 (previously known as ADCT-401) is an ADC consisting of an engineered version of the anti-PSMA antibody J591 site specifically conjugated to the pyrrolobenzodiazepine (PBD) dimer tesirine. MEDI3726 specifically binds the extracellular domain of PSMA and, once internalized, releases the PBD dimer to crosslink DNA and trigger cell death. In vitro, MEDI3726 demonstrated potent and specific cytotoxicity in a panel of PSMA-positive prostate cancer cell lines, consistent with internalization and DNA interstrand crosslinking. In vivo, MEDI3726 showed robust antitumor activity against the LNCaP and the castration-resistant CWR22Rv1 prostate cancer cell line xenografts. MEDI3726 also demonstrated durable antitumor activity in the PSMA-positive human prostate cancer patient–derived xenograft (PDX) LuCaP models. This activity correlated with increased phosphorylated Histone H2AX in tumor xenografts treated with MEDI3726. MEDI3726 is being evaluated in a phase I clinical trial as a treatment for patients with metastatic castrate-resistant prostate cancer (NCT02991911). Mol Cancer Ther; 17(10); 2176–86. ©2018 AACR.

Abstract A004: MEDI3726 (ADCT-401), a novel antibody-drug conjugate targeting PSMA, has potent in vivo antitumor activity in prostate cancer patient-derived xenograft models

Prostate specific membrane antigen (PSMA), a type II membrane glycoprotein, is highly expressed in nearly all prostate cancers, with the highest expression in metastatic castration-resistant prostate cancer (mCRPC). The prevalence, increased surface expression in prostate tumors, and constitutive internalization make PSMA an attractive target for an antibody-drug conjugate (ADC) approach in anticancer treatment of patients with mCRPC. MEDI3726 (previously known as ADCT-401) is an ADC consisting of an engineered version of anti-PSMA antibody J591, site-specifically conjugated with DNA cross-linking pyrrolobenzodiazepine (PBD) dimer SG3249 for targeting prostatic cancer cells. Using prostate cancer cell line models, we have previously shown that MEDI3726 specifically binds to the extracellular domain of PSMA and, once internalized, releases the PBD dimer to cross link DNA and achieve potent in vitro and in vivo cytotoxicity. Here we investigated the in vivo activity of MEDI3726 in a series of LuCaP prostate cancer patient-derived xenograft (PDX) models. The selected LuCaP models had varying PSMA expression and heterogeneous genetic and phenotypic backgrounds. In agreement with the earlier cell line xenograft data, dose-dependent antitumor activity was observed in PSMA-positive PDX models with durable tumor regressions in models with high PSMA expression. In the PSMA-negative LuCaP 35CR PDX model, MEDI3726 did not have significant antitumor activity, thus highlighting target-mediated in vivo activity. Increased phosphorylation of histone H2AX was observed in xenografts dosed with MEDI3726, confirming DNA damage induced by interstrand cross-linking PBD dimer as the mechanism of antitumor activity of MEDI3726. In summary, MEDI3726 demonstrated potent and specific in vivo antitumor activity, concurrent with DNA damage, in clinically relevant prostate cancer PDX models. MEDI3726 is being evaluated in phase 1 clinical trial as an anticancer treatment in patients with metastatic castrate-resistant prostate cancer (NCT02991911). Citation Format: Song Cho, Francesca Zammarchi, Noel R. Monks, Kapil Vashisht, Ravinder Tammali, Kevin Schifferli, Patrick Strout, Wanda King, Karma Dacosta, Ryan Fleming, David G. Williams, Karin Havenith, Mary Jane Masson Hinrichs, Simon Chivers, Nazzareno Dimasi, Phil W. Howard, John A. Hartley, Steve Coats, Ronald Herbst, Patrick H. van Berkel, David A. Tice. MEDI3726 (ADCT-401), a novel antibody-drug conjugate targeting PSMA, has potent in vivo antitumor activity in prostate cancer patient-derived xenograft models [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr A004.

Abstract LB-295: MEDI7247, a novel pyrrolobenzodiazepine ADC targeting ASCT2 with potent in vivo activity across a spectrum of hematological malignancies

MEDI7247 is a first in class ADC consisting of a human anti-ASCT2 monoclonal antibody site specifically conjugated to DNA cross-linking pyrrolobenzodiazepine (PBD) dimers. ASCT2 (SLC1A5) is a multi-pass, Na+-dependent neutral amino acid transporter that mediates the uptake of amino acids required for tumor growth and progression. ASCT2 is highly overexpressed in many hematologic cancers, most notably Multiple Myeloma (MM - 100% positive), Acute Myeloid Leukemia (AML - 100% positive) and Diffuse Large B cell lymphoma (DLBCL - 95% positive). ASCT2 expression is low in normal tissues. MEDI7247 (Q1Wx4) demonstrated a significant survival advantage in 3 disseminated AML cell line models, TF1α(ASCT2-High), MOLM-13(ASCT2-low) and M.V.411(ASCT2-High), when compared to the untreated control at the lowest dose levels examined: 0.05, 0.1 and 0.1 mg/kg, respectively. Further exemplifying the activity of MEDI7247, both the TF1α and MOLM-13 models did not reach 50% survival by the end of the study, with 80% survival at >200 days for TF1α and 70% survival at >180 days for MOLM-13. Similarly, a single dose of MEDI7247 in the TF1α model resulted in a 60% survival at >200 days at 0.05 mg/kg. MEDI7247 was also tested in a disseminated AML PDX(ASCT2-low) model at 0.05, 0.1 and 0.4 mg/kg. A significant improvement in survival was observed at both 0.1 and 0.4 mg/kg with the higher dose level extending survival by >80 days. MEDI7247 activity was further confirmed by monitoring peripheral blood CD33+ve cells, which initially receded, with the timing of reappearance preempting survival. Multiple Myeloma is another indication that exhibits a high level of ASCT2 expression. MEDI7247 (Q1Wx4) efficacy was examined in 3 disseminated MM cell line models, NCI-H929(ASCT2-High), MM.1S(ASCT2-medium) and OPM2(ASCT2-Medium), with a significant improvement in survival from control at the lowest dose levels examined: 0.1, 0.1 and 0.05 mg/kg, respectively. The activity of MEDI7247 (Q1Wx4) was also examined in the subcutaneous DLBCL model KARPAS 422(ASCT2-High). Tumor regressions were observed at all dose levels tested (0.1, 0.2, 0.3 and 0.4 mg/kg), with the higher two dose levels resulting in complete tumor regression without regrowth beyond 150 days. Additionally, MEDI7247 (Q1Wx4) is efficacious against the disseminated 697(ASCT2-Low) (Acute Lymphoblastic Leukemia - ALL) and RAJI(ASCT2-High) (Burkitt9s lymphoma) models. A significant survival advantage was seen in both tumor models at the lowest dose examined of 0.05 mg/kg. In conclusion, MEDI7247 demonstrates antitumor efficacy across all tumor indications tested and varying levels of ASCT2 expression. These data support the use of MEDI7247 in ASCT2 positive hematological malignancies. MEDI7247 is currently in Phase 1 clinical trials. Citation Format: Noel R. Monks, Kevin P. Schifferli, Ravinder Tammali, M. Jack Borrok, Steven R. Coats, Ronald Herbst, David A. Tice, Nabendu Pore. MEDI7247, a novel pyrrolobenzodiazepine ADC targeting ASCT2 with potent in vivo activity across a spectrum of hematological malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-295.

Abstract 76: Synthetic lethal targeting ofBRCAmutant tumors with antibody linked pyrrolobenzodiazepine dimers

Pyrrolbenzodiazepine dimers (PBDs) are amongst the most potent DNA alkylating agents, with activity against a broad spectrum of tumors. PBDs form cross-links within the minor groove of DNA causing double strand breaks (DSB). DNA repair genes such as BRCA1 and BRCA2 play important roles in homologous recombination repair (HRR) of DSB. Cells defective in BRCA1 or BRCA2 are known to be sensitive to DNA interstrand crosslinks. Accordingly, it is possible that PBD-based ADCs will have enhanced killing of cells (synthetic lethality) in which HR processes are defective by inactivation of BRCA1 or BRCA2 genes in breast, ovarian and other cancers. To determine anti-tumor activity of PBD dimers, we have used MEDI0641, PBD-dimer conjugated to anti-5T4 antibody, against BRCA wild type and mutant xenograft tumor models. MEDI0641 was >3-fold more potent in BRCA1 or BRCA2 mutant models than in wild-type xenografts. Similar observations were seen in 25 patient-derived xenograft (PDX) models (19 breast and 6 ovarian) bearing mutations in BRCA1 or BRCA2 (blinded to 5T4 expression) treated with MEDI0641. Out of a total of 25 PDX models, 17 models had tumor regression with a single administration of MEDI0641 at 0.3 mg/kg (response rate = 68%), and 14 models showed response to 0.1 mg/kg of MEDI0641 (response rate = 56%). In BRCA wild-type PDX models, a higher dose of 1 mg/kg was required to achieve full anti-tumor efficacy. Retrospective analysis of 5T4 expression in PDX tumors demonstrated no correlation between efficacy and target expression in BRCA mutant PDX models. To further delineate the role of BRCA1/2 mutations in determining sensitivity to PBD, we used siRNA knock-down of both BRCA1 and BRCA2 in the DNA repair wild type HeLa cells. Knockdown of BRCA genes sensitized Hela cells to PBD payload and MEDI0641 in vitro. Anti-tumor activity of MEDI0641 was further examined in isogenic BRCA2 knockout xenograft models. Genetic deletion of BRCA2 markedly increased anti-tumor activity of MEDI0641. In conclusion, PBD based ADCs may have improved therapeutic window in cancer patients with somatic BRCA mutations. Citation Format: Haihong Zhong, Ravinder Tammali, Cui Chen, Christine Fazenbaker, Kennedy Maureen, Noel Monks, Jay Harper, Ronald Herbst, Dave Tice. Synthetic lethal targeting of BRCA mutant tumors with antibody linked pyrrolobenzodiazepine dimers [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 76. doi:10.1158/1538-7445.AM2017-76