Epidermal growth factor receptor (EGFR) and HER3 each form heterodimers with HER2 and have independently been implicated as key coreceptors that drive HER2-amplified breast cancer. Some studies suggest a dominant role for EGFR, a notion of renewed interest given the development of dual HER2/EGFR small-molecule inhibitors. Other studies point to HER3 as the primary coreceptor. To clarify the relative contributions of EGFR and HER3 to HER2 signaling, we studied receptor knockdown via small interfering RNA technology across a panel of six HER2-overexpressing cell lines. Interestingly, HER3 was as critical as HER2 for maintaining cell proliferation in most cell lines, whereas EGFR was dispensable. Induction of HER3 knockdown in the HER2-overexpressing BT474M1 cell line was found to inhibit growth in three-dimensional culture and induce rapid tumor regression of in vivo xenografts. Furthermore, preferential phosphorylation of HER3, but not EGFR, was observed in HER2-amplified breast cancer tissues. Given these data suggesting HER3 as an important therapeutic target, we examined the activity of pertuzumab, a HER2 antibody that inhibits HER3 signaling by blocking ligand-induced HER2/HER3 heterodimerization. Pertuzumab inhibited ligand-dependent morphogenesis in three-dimensional culture and induced tumor regression in the heregulin-dependent MDA-MB-175 xenograft model. Importantly, these activities of pertuzumab were distinct from those of trastuzumab, a monoclonal antibody currently used for treatment of HER2-amplified breast cancer patients. Our data suggest that inhibition of HER3 may be more clinically relevant than inhibition of EGFR in HER2-amplified breast cancer and also suggest that adding pertuzumab to trastuzumab may augment therapeutic benefit by blocking HER2/ HER3 signaling.

The growth and morphological differentiation of dendrites are critical events in the establishment of proper neuronal connectivity and neural function. One extrinsic factor, BMP7, has been shown to specifically affect dendritic morphogenesis; however, the underlying mechanism by which this occurs is unknown. Here we show that LIM kinase 1 (LIMK1), a key downstream effector of Rho GTPases, colocalizes with the BMP receptor, BMPRII, in the tips of neurites and binds to BMPRII. This interaction is required for BMP-dependent induction of the dendritic arbor in cortical neurons. Furthermore, we demonstrate that the physical interaction of LIMK1 with BMPRII synergizes with the Rho GTPase, Cdc42, to activate LIMK1 catalytic activity. These studies thus define a Smad-independent pathway that directly links the BMP receptor to regulation of actin dynamics and provides insights into how extracellular signals modulate LIMK1 activity to permit fine spatial control over cytoskeletal remodelling during dendritogenesis.

SummaryThe large transforming growth factor-β (TGFβ) superfamily of secreted proteins regulate the growth, development and differentiation of cells in diverse organisms, including nematode worms, flies, mice and humans. Signals are initiated upon binding of TGFβ superfamily members to cell-surface serine/threonine kinase receptors and are then propagated by the intracellular mediators known as Smads. Activation of Smads results in their translocation from the cytoplasm into the nucleus, where they activate or repress transcription together with transcription factors so as to regulate target gene expression. Most Smads consist of two conserved domains, Mad homology (MH) domains 1 and 2, which are separated by a non-conserved linker region. These domains lack enzymatic activity and, instead, Smads mediate their effects through protein-protein and protein-DNA interactions. Targeted disruption of Smad genes in mice has revealed their importance in embryonic development, and a tumor-suppressor role for Smads in human cancers has been described. Smads therefore play an essential role in mediating TGFβ-superfamily signals in development and disease.

Purpose: Oncogenic activation of the phosphatidylinositol 3-kinase (PI3K) signaling pathway is prevalent in breast cancer and has been associated with resistance to HER2 inhibitors in the clinic.We therefore investigated the combinatorial activity of GDC-0941, a novel class IPI3K inhibitor, with standard-of-care therapies for HER2-amplified breast cancer. Experimental Design: Three-dimensional laminin-rich extracellular matrix cultures of human breast cancer cells were utilized to provide a physiologically relevant approach to analyze the efficacy and molecular mechanism of combination therapies ex vivo. Combination studies were done using GDC-0941 with trastuzumab (Herceptin), pertuzumab, lapatinib (Tykerb), and docetaxel, the principal therapeutic agents that are either approved or being evaluated for treatment of early HER2-positive breast cancer.Results: Significant GDC-0941 activity (EC 50 <1 Amol/L) was observed for >70% of breast cancer cell lines that were examined in three-dimensional laminin-rich extracellular matrix culture. Differential responsiveness to GDC-0941as a single agent was observed for luminal breast cancer cells upon stimulation with the HER3 ligand, heregulin. Combined treatment of GDC-0941, trastuzumab, and pertuzumab resulted in growth inhibition, altered acinar morphology, and suppression of AKT mitogen-activated protein kinase (MAPK) / extracellular signed-regulated kinase (ERK) kinase and MEK effector signaling pathways for HER2-amplified cells in both normal and heregulin-supplemented media. The GDC-0941 and lapatinib combination further showed that inhibition of HER2 activity was essential for maximum combinatorial efficacy. PI3K inhibition also rendered HER2-amplified BT-474M1cells and tumor xenografts more sensitive to docetaxel. Conclusions: GDC-0941 is efficacious in preclinical models of breast cancer. The addition of GDC-0941to HER2-directed treatment could augment clinical benefit in breast cancer patients.

c-KIT overexpression is well recognized in cancers such as gastrointestinal stromal tumors (GIST), small cell lung cancer (SCLC), melanoma, non-small cell lung cancer (NSCLC), and acute myelogenous leukemia (AML). Treatment with the small-molecule inhibitors imatinib, sunitinib, and regorafenib resulted in resistance (c-KIT mutant tumors) or limited activity (c-KIT wild-type tumors). We selected an anti-c-KIT ADC approach to evaluate the anticancer activity in multiple disease models. A humanized anti-c-KIT antibody LMJ729 was conjugated to the microtubule destabilizing maytansinoid, DM1, via a noncleavable linker (SMCC). The activity of the resulting ADC, LOP628, was evaluated against GIST, SCLC, and AML models and against GIST and SCLC models. LOP628 exhibited potent antiproliferative activity on c-KIT-positive cell lines, whereas LMJ729 displayed little to no effect. At exposures predicted to be clinically achievable, LOP628 demonstrated single administration regressions or stasis in GIST and SCLC xenograft models in mice. LOP628 also displayed superior efficacy in an imatinib-resistant GIST model. Further, LOP628 was well tolerated in monkeys with an adequate therapeutic index several fold above efficacious exposures. Safety findings were consistent with the pharmacodynamic effect of neutropenia due to c-KIT-directed targeting. Additional toxicities were considered off-target and were consistent with DM1, such as effects in the liver and hematopoietic/lymphatic system. The preclinical findings suggest that the c-KIT-directed ADC may be a promising therapeutic for the treatment of mutant and wild-type c-KIT-positive cancers and supported the clinical evaluation of LOP628 in GIST, AML, and SCLC patients. .