Transcriptional Subtypes Resolve Tumor Heterogeneity and Identify Vulnerabilities to MEK Inhibition in Lung Adenocarcinoma.

PURPOSE: Lung adenocarcinomas comprise the largest fraction of non-small cell lung cancer, which is the leading cause of cancer-related deaths. Seventy-five percent of adenocarcinomas lack targeted therapies because of scarcity of druggable drivers. Here, we classified tumors on the basis of signaling similarities and discovered subgroups within this unmet patient population. EXPERIMENTAL DESIGN: We leveraged transcriptional data from >800 early- and advanced-stage patients. RESULTS: We identified three robust subtypes dubbed mucinous, proliferative, and mesenchymal with respective pathway phenotypes. These transcriptional states lack discrete and causative mutational etiology as evidenced by similarly distributed oncogenic drivers, including KRAS and EGFR. The subtypes capture heterogeneity even among tumors lacking known oncogenic drivers. Paired multi-regional intratumoral biopsies demonstrated unified subtypes despite divergently evolved prooncogenic mutations, indicating subtype stability during selective pressure. Heterogeneity among in vitro and in vivo preclinical models is expounded by the human lung adenocarcinoma subtypes and can be leveraged to discover subtype-specific vulnerabilities. As proof of concept, we identified differential subtype response to MEK pathway inhibition in a chemical library screen of 89 lung cancer cell lines, which reproduces across model systems and a clinical trial. CONCLUSIONS: Our findings support forward translational relevance of transcriptional subtypes, where further exploration therein may improve lung adenocarcinoma treatment.See related commentary by Skoulidis, p. 913.

Combination of mAb-AR20.5, anti-PD-L1 and PolyICLC inhibits tumor progression and prolongs survival of MUC1.Tg mice challenged with pancreatic tumors.

A substantial body of evidence suggests the existence of MUC1-specific antibodies and cytotoxic T cell activities in pancreatic cancer patients. However, tumor-induced immunosuppression renders these responses ineffective. The current study explores a novel therapeutic combination wherein tumor-bearing hosts can be immunologically primed with their own antigen, through opsonization with a tumor antigen-targeted antibody, mAb-AR20.5. We evaluated the efficacy of immunization with this antibody in combination with PolyICLC and anti-PD-L1. The therapeutic combination of mAb-AR20.5 + anti-PD-L1 + PolyICLC induced rejection of human MUC1 expressing tumors and provided a long-lasting, MUC1-specific cellular immune response, which could be adoptively transferred and shown to provide protection against tumor challenge in human MUC1 transgenic (MUC.Tg) mice. Furthermore, antibody depletion studies revealed that CD8 cells were effectors for the MUC1-specific immune response generated by the mAb-AR20.5 + anti-PD-L1 + PolyICLC combination. Multichromatic flow cytometry data analysis demonstrated a significant increase over time in circulating, activated CD8 T cells, CD3+CD4-CD8-(DN) T cells, and mature dendritic cells in mAb-AR20.5 + anti-PD-L1 + PolyICLC combination-treated, tumor-bearing mice, as compared to saline-treated control counterparts. Our study provides a proof of principle that an effective and long-lasting anti-tumor cellular immunity can be achieved in pancreatic tumor-bearing hosts against their own antigen (MUC1), which can be further potentiated using a vaccine adjuvant and an immune checkpoint inhibitor.

Targeting PTPRK-RSPO3 colon tumours promotes differentiation and loss of stem-cell function.

Colorectal cancer remains a major unmet medical need, prompting large-scale genomics efforts in the field to identify molecular drivers for which targeted therapies might be developed. We previously reported the identification of recurrent translocations in R-spondin genes present in a subset of colorectal tumours. Here we show that targeting RSPO3 in PTPRK-RSPO3-fusion-positive human tumour xenografts inhibits tumour growth and promotes differentiation. Notably, genes expressed in the stem-cell compartment of the intestine were among those most sensitive to anti-RSPO3 treatment. This observation, combined with functional assays, suggests that a stem-cell compartment drives PTPRK-RSPO3 colorectal tumour growth and indicates that the therapeutic targeting of stem-cell properties within tumours may be a clinically relevant approach for the treatment of colorectal tumours.

Redesigning a Monospecific Anti-FGFR3 Antibody to Add Selectivity for FGFR2 and Expand Antitumor Activity.

FGF receptors (FGFR) are attractive candidate targets for cancer therapy because they are dysregulated in several human malignancies. FGFR2 and FGFR3 can be inhibited potentially without disrupting adult tissue homeostasis. In contrast, blocking the closely related FGFR1 and FGFR4, which regulate specific metabolic functions, carries a greater safety risk. An anti-FGFR3 antibody was redesigned here to create function-blocking antibodies that bind with dual specificity to FGFR3 and FGFR2 but spare FGFR1 and FGFR4. R3Mab, a previously developed monospecific anti-FGFR3 antibody, was modified via structure-guided phage display and acquired additional binding to FGFR2. The initial variant was trispecific, binding tightly to FGFR3 and FGFR2 and moderately to FGFR4, while sparing FGFR1. The X-ray crystallographic structure indicated that the antibody variant was bound to a similar epitope on FGFR2 as R3Mab on FGFR3. The antibody was further engineered to decrease FGFR4-binding affinity while retaining affinity for FGFR3 and FGFR2. The resulting dual-specific antibodies blocked FGF binding to FGFR3 and FGFR2 and inhibited downstream signaling. Moreover, they displayed efficacy in mice against human tumor xenografts overexpressing FGFR3 or FGFR2. Thus, a monospecific antibody can be exquisitely tailored to confer or remove binding to closely related targets to expand and refine therapeutic potential.

An Antibody-Drug Conjugate Directed against Lymphocyte Antigen 6 Complex, Locus E (LY6E) Provides Robust Tumor Killing in a Wide Range of Solid Tumor Malignancies.

PURPOSE: Chemotherapies are limited by a narrow therapeutic index resulting in suboptimal exposure of the tumor to the drug and acquired tumor resistance. One approach to overcome this is through antibody-drug conjugates (ADC) that facilitate greater potency via target-specific delivery of highly potent cytotoxic agents. EXPERIMENTAL DESIGN: In this study, we used a bioinformatics approach to identify the lymphocyte antigen 6 complex locus E (LY6E), an IFN-inducible glycosylphosphatidylinositol (GPI)-linked cell membrane protein as a promising ADC target. We developed a monoclonal anti-LY6E antibody and characterized in situ LY6E expression in over 750 cancer specimens and normal tissues. Target-dependent anti-LY6E ADC killing was investigated both in vitro and in vivo using patient-derived xenograft models. RESULTS: Using in silico approaches, we found that LY6E was significantly overexpressed and amplified in a wide array of different human solid tumors. IHC analysis revealed high LY6E protein expression in a number of tumor types, such as breast, lung, gastric, ovarian, pancreatic, kidney and head/neck carcinomas. Characterization of the endocytic pathways for LY6E revealed that the LY6E-specific antibody is internalized into cells leading to lysosomal accumulation. Consistent with this, a LY6E-specific ADC inhibited in vitro cell proliferation and produced durable tumor regression in vivo in clinically relevant LY6E-expressing xenograft models. CONCLUSIONS: Our results identify LY6E as a highly promising molecular ADC target for a variety of solid tumor types with current unmet medical need.

An anti-B7-H4 antibody-drug conjugate for the treatment of breast cancer.

B7-H4 has been implicated in cancers of the female reproductive system and investigated for its possible use as a biomarker for cancer, but there are no preclinical studies to demonstrate that B7-H4 is a molecular target for therapeutic intervention of cancer. We provide evidence that the prevalence and expression levels of B7-H4 are high in different subtypes of breast cancer and that only a few normal tissues express B7-H4 on the cell membrane. These profiles of low normal expression and upregulation in cancer provide an opportunity for the use of antibody-drug conjugates (ADCs), cytotoxic drugs chemically linked to antibodies, for the treatment of B7-H4 positive cancers. We have developed an ADC specific to B7-H4 that uses a linker drug consisting of a potent antimitotic, monomethyl auristatin E (MMAE), linked to engineered cysteines (THIOMAB) via a protease labile linker. We will refer to ADCs that use the THIOMAB format as TDCs to help distinguish the format from standard MC-vc-MMAE ADCs that are conjugated to the interchain disulfide bonds. Anti-B7-H4 (h1D11)-MC-vc-PAB-MMAE (h1D11 TDC) produced durable tumor regression in cell line and patient-derived xenograft models of triple-negative breast cancer. It also binds rat B7-H4 with similar affinity to human and allowed us to test for target dependent toxicity in rats. We found that our anti-B7-H4 TDC has toxicity findings similar to untargeted TDC. Our results validate B7-H4 as an ADC target for breast cancer and support the possible use of this TDC in the treatment of B7-H4(+) breast cancer.

Interactions between MUC1 and p120 catenin regulate dynamic features of cell adhesion, motility, and metastasis.

The mechanisms by which MUC1 and p120 catenin contribute to progression of cancers from early transformation to metastasis are poorly understood. Here we show that p120 catenin ARM domains 1, 3-5, and 8 mediate interactions between p120 catenin and MUC1, and that these interactions modulate dynamic properties of cell adhesion, motility, and metastasis of pancreatic cancer cells. We also show that different isoforms of p120 catenin, when coexpressed with MUC1, create cells that exhibit distinct patterns of motility in culture (motility independent of cell adhesion, motility within a monolayer while exchanging contacts with other cells, and unified motility while maintaining static epithelial contacts) and patterns of metastasis. The results provide new insight into the dynamic interplay between cell adhesion and motility and the relationship of these to the metastatic process.

Dependence of tumor cell lines and patient-derived tumors on the NAD salvage pathway renders them sensitive to NAMPT inhibition with GNE-618.

Nicotinamide adenine dinucleotide (NAD) is a critical metabolite that is required for a range of cellular reactions. A key enzyme in the NAD salvage pathway is nicotinamide phosphoribosyl transferase (NAMPT), and here, we describe GNE-618, an NAMPT inhibitor that depletes NAD and induces cell death in vitro and in vivo. While cells proficient for nicotinic acid phosphoribosyl transferase (NAPRT1) can be protected from NAMPT inhibition as they convert nicotinic acid (NA) to NAD independent of the salvage pathway, this protection only occurs if NA is added before NAD depletion. We also demonstrate that tumor cells are unable to generate NAD by de novo synthesis as they lack expression of key enzymes in this pathway, thus providing a mechanistic rationale for the reliance of tumor cells on the NAD salvage pathway. Identifying tumors that are sensitive to NAMPT inhibition is one potential way to enhance the therapeutic effectiveness of an NAMPT inhibitor, and here, we show that NAMPT, but not NAPRT1, mRNA and protein levels inversely correlate with sensitivity to GNE-618 across a panel of 53 non-small cell lung carcinoma cell lines. Finally, we demonstrate that GNE-618 reduced tumor growth in a patient-derived model, which is thought to more closely represent heterogeneous primary patient tumors. Thus, we show that dependence of tumor cells on the NAD salvage pathway renders them sensitive to GNE-618 in vitro and in vivo, and our data support further evaluation of the use of NAMPT mRNA and protein levels as predictors of overall sensitivity.

Identification of amides derived from 1H-pyrazolo[3,4-b]pyridine-5-carboxylic acid as potent inhibitors of human nicotinamide phosphoribosyltransferase (NAMPT).

Potent, 1H-pyrazolo[3,4-b]pyridine-containing inhibitors of the human nicotinamide phosphoribosyltransferase (NAMPT) enzyme were identified using structure-based design techniques. Many of these compounds exhibited nanomolar antiproliferation activities against human tumor lines in in vitro cell culture experiments, and a representative example (compound 26) demonstrated encouraging in vivo efficacy in a mouse xenograft tumor model derived from the A2780 cell line. This molecule also exhibited reduced rat retinal exposures relative to a previously studied imidazo-pyridine-containing NAMPT inhibitor. Somewhat surprisingly, compound 26 was only weakly active in vitro against mouse and monkey tumor cell lines even though it was a potent inhibitor of NAMPT enzymes derived from these species. The compound also exhibited only minimal effects on in vivo NAD levels in mice, and these changes were considerably less profound than those produced by an imidazo-pyridine-containing NAMPT inhibitor. The crystal structures of compound 26 and the corresponding PRPP-derived ribose adduct in complex with NAMPT were also obtained.

P21-activated kinase 1 (PAK1) as a therapeutic target in BRAF wild-type melanoma.

BACKGROUND: Although remarkable clinical response rates in melanoma have been observed using vemurafenib or dabrafenib in patients with tumors carrying oncogenic mutations in BRAF, a substantial unmet medical need remains for the subset of patients with wild-type BRAF tumors. METHODS: To investigate the role of p21-activated kinases (PAKs) in melanoma, we determined PAK1 genomic copy number and protein expression for a panel of human melanoma tissues. PAK1 was inhibited in vitro and in vivo using RNA interference or PF-3758309 inhibitor treatment in a panel of melanoma cell lines with known BRAF and RAS (rat sarcoma) genotype to better understand its role in melanoma cell proliferation and migration. Tumorigenesis was assessed in vivo in female NCR nude mice and analyzed with cubic spline regression and area under the curve analyses. All statistical tests were two-sided. RESULTS: Strong cytoplasmic PAK1 protein expression was prevalent in melanomas (27%) and negatively associated with activating mutation of the BRAF oncogene (P < .001). Focal copy number gain of PAK1 at 11q13 was also observed in 9% of melanomas (n = 87; copy number ≥ 2.5) and was mutually exclusive with BRAF mutation (P < .005). Selective PAK1 inhibition attenuated signaling through mitogen-activated protein kinase (MAPK) as well as cytoskeleton-regulating pathways to modulate the proliferation and migration of BRAF wild-type melanoma cells. Treatment of BRAF wild-type melanomas with PF-3758309 PAK inhibitor decreased tumor growth for SK-MEL23 and 537MEL xenografts (91% and 63% inhibition, respectively; P < .001) and MAPK pathway activation in vivo. CONCLUSIONS: Taken together, our results provide evidence for a functional role of PAK1 in BRAF wild-type melanoma and therapeutic use of PAK inhibitors in this indication.

mTOR kinase inhibitor potentiates apoptosis of PI3K and MEK inhibitors in diagnostically defined subpopulations.

The mammalian target of rapamycin (mTOR) is a central node in a complex signaling network that is regulated by several pathways deregulated in human cancers, including the PI3K/Akt and MAPK pathways. Targeting mTOR therefore presents an opportunity for therapeutic intervention. However, mTOR inhibition with rapamycin analogs or kinase inhibitors reduces cell growth but does not induce apoptosis, and the clinical benefit of rapamycin analogs has been modest. In this study we show that mTOR kinase inhibitors can potentiate apoptosis when used in combination with upstream targeted agents such as PI3K and MEK inhibitors. This increased apoptosis is dependent on genetic background, and correlates with active growth factor survival pathways. In PI3K mutant tumors, mTOR inhibition leads to partial reactivation of Akt which allows cells to survive, whereas in KRAS mutant tumors, this same reactivation of Akt occurs but is not required for cell survival. These data suggest the use of selected rational combinations of mTOR kinase inhibitors with other targeted inhibitors in specific tumor genotypes to achieve the maximal cytotoxic response by inhibiting two nodes in the activated signaling network.

Cyclin-dependent kinase 5 is amplified and overexpressed in pancreatic cancer and activated by mutant K-Ras.

PURPOSE: To evaluate the nature of cyclin-dependent kinase 5 (CDK5) hyperactivity in pancreatic cancer progression. EXPERIMENTAL DESIGN: We used genetic, biochemical, and molecular biology methods to investigate the nature and function of overexpression of CDK5 and its activators p35 and p39 during the progression of pancreatic cancer. RESULTS: Amplification of the CDK5 gene or either of its main activators, p35 and p39, was observed in 67% of human pancreatic ductal adenocarcinoma (PDAC). CDK5, p35, and p39 were rarely expressed in pancreatic ducts whereas more than 90% of PDACs had increased levels of CDK5 and p35. Increased levels of CDK5, p35, and p39 protein were observed in several pancreatic cancer cell lines. Inhibition of CDK5 kinase activity using a CDK5 dominant-negative mutant or the drug roscovitine significantly decreased the migration and invasion of pancreatic cancer cells in vitro. Increased CDK5 kinase activity was also observed in immortalized human pancreatic nestin-expressing (HPNE) cells expressing a mutant form of K-Ras (G12D) compared with HPNE cells expressing native K-Ras. G12D K-Ras increased cleavage of p35 to p25, a stable and greater activator of CDK5, thus implicating a role for CDK5 in early progression of PDAC. Inhibition of the signaling cascade downstream of mutant K-Ras (G12D) that involves mitogen-activated protein/extracellular signal-regulated kinase, phosphoinositide 3-kinase, or CDK5 decreased p25 protein levels. CONCLUSION: These results suggest that mutant K-Ras acts in concert with CDK5 and its activators to increase malignant progression, migration, and invasion of pancreatic cancer cells.

Flt3 ligand delivered in a pluronic formulation prolongs the survival of mice with orthotopic pancreatic adenocarcinoma.

Pancreatic adenocarcinoma is a devastating disease, characterized by asymptomatic development and extremely poor prognosis. Given the resistance of pancreatic cancer to standard chemo- and radiotherapy, we have focused on the development of immunotherapies for this disease. The number of dendritic cells (DCs), natural killer (NK) cells, and T-cells in the blood and secondary lymphoid organs is regulated by a group of hematopoietic growth factors, which includes fms-like tyrosine kinase-3 ligand (Flt3L). We have demonstrated previously that the bioavailability and in vivo half-life of Flt3L are increased by Flt3L formulation in the pluronic ProGelzx. In this study, we first examined the effectiveness of Flt3L delivered in ProGelz against subcutaneous (s.c.) pancreatic adenocarcinomas in mice. We found that an intramuscular (i.m.) injection of Flt3L in ProGelz significantly increased the survival of mice bearing s.c. pancreatic tumors, compared to the administration of phosphate-buffered saline (PBS) in ProGelz. We then tested Flt3L in ProGelz in an orthotopic pancreatic tumor model, and demonstrated that it significantly enhanced the survival of tumor-bearing mice, compared to PBS in ProGelz. Overall, these observations suggest that Flt3L formulated in ProGelz may have potential clinical utility as a treatment for pancreatic cancer.