PDZ-RhoGEF and LARG are essential for embryonic development and provide a link between thrombin and LPA receptors and Rho activation.

G protein-coupled receptors (GPCRs) linked to both members of the Gα12 family of heterotrimeric G proteins α subunits, Gα12 and Gα13, regulate the activation of Rho GTPases, thereby contributing to many key biological processes. Multiple Rho GEFs have been proposed to link Gα12/13 GPCRs to Rho activation, including PDZ-RhoGEF (PRG), leukemia-associated Rho GEF (LARG), p115-RhoGEF (p115), lymphoid blast crisis (Lbc), and Dbl. PRG, LARG, and p115 share the presence of a regulator of G protein signaling homology (RGS) domain. There is limited information on the biological roles of this RGS-containing family of RhoGEFs in vivo. p115-deficient mice are viable with some defects in the immune system and gastrointestinal motor dysfunctions, whereas in an initial study we showed that mice deficient for Larg are viable and resistant to salt-induced hypertension. Here, we generated knock-out mice for Prg and observed that these mice do not display any overt phenotype. However, deficiency in Prg and Larg leads to complex developmental defects and early embryonic lethality. Signaling from Gα11/q-linked GPCRs to Rho was not impaired in mouse embryonic fibroblasts defective in all three RGS-containing RhoGEFs. However, a combined lack of Prg, Larg, and p115 expression abolished signaling through Gα12/13 to Rho and thrombin-induced cell proliferation, directional migration, and nuclear signaling through JNK and p38. These findings provide evidence of an essential role for the RGS-containing RhoGEF family in signaling to Rho by Gα12/13-coupled GPCRs, which may likely play a critical role during embryonic development.

An essential role for Rac1 in endothelial cell function and vascular development.

Numerous cell surface receptors, including tyrosine kinase and G protein-coupled receptors, play critical roles in endothelial cell function and blood vessel development. These receptors share the ability of stimulating an intricate network of intracellular signaling pathways, including the activation of members of the Ras and Rho family of small GTPases. However, the contribution of these signaling molecules to the numerous biological activities performed by endothelial cells is still not fully understood. Here, we have used a conditional Cre/Flox approach, enabling the deletion of the Rac1 gene in endothelial cells, to examine the role of the Rho-related GTPase Rac1 in endothelial cell function and vascular development. Rac1 excision in primary endothelial cells in vitro revealed that Rac1 plays a central role in endothelial cell migration, tubulogenesis, adhesion, and permeability in response to vascular endothelial growth factor (VEGF) and sphingosine-1-phosphate (S1P), which is likely due to the inability of Rac1-deficient endothelial cells to form lamellipodial structures and focal adhesions, and to remodel their cell-cell contacts. Importantly, endothelial-specific excision of Rac1 results in embryonic lethality in midgestation (around E9.5), and defective development of major vessels and complete lack of small branched vessels was readily observed in these endothelial Rac1-deficient embryos and their yolk sacs. These findings provide direct evidence that the activity of Rac1 in endothelial cells is essential for vascular development and suggest that Rac1 and its downstream targets may represent promising therapeutic targets for the treatment of numerous human diseases that involve aberrant neovascularization.

Multiple PPPS/TP motifs act in a combinatorial fashion to transduce Wnt signaling through LRP6.

Binding of Wnt to Frizzled, and either of two members of the low-density-lipoprotein receptor-related protein family, LRP5/6, leads to beta-catenin activation by a poorly understood mechanism. LRP5/6 exhibit five highly conserved PPPS/TP motifs in their intracellular region, among which the first PPPS/TP site is rapidly phosphorylated upon Wnt stimulation. By the use of full-length LRP6 mutants harboring multiple mutations involving the five PPPS/TP motifs, we found that this first PPPS/TP phosphoacceptor site is alone not sufficient or strictly necessary for beta-catenin activation. Instead, we show that each LRP6 PPPS/TP motif contributes in a combinatorial fashion to activate the canonical Wnt-beta-catenin pathway.

U.S. Food and Drug Administration Approval: Neratinib for the Extended Adjuvant Treatment of Early-Stage HER2-Positive Breast Cancer.

On July 17, 2017, the FDA approved neratinib (NERLYNX; Puma Biotechnology, Inc.) for the extended adjuvant treatment of adult patients with early-stage HER2-overexpressed/amplified breast cancer, to follow adjuvant trastuzumab-based therapy. Approval was based on data from ExteNET, a randomized, double-blind, placebo-controlled multicenter trial. Women with early-stage HER2-positive breast cancer and within 2 years of completing adjuvant trastuzumab were randomized to neratinib (n = 1,420) or placebo (n = 1,420) for 1 year. The primary endpoint was invasive disease-free survival (iDFS), defined as the time between randomization date to first occurrence of invasive recurrence (local/regional, ipsilateral, or contralateral breast cancer), distant recurrence, or death from any cause, with 2 years and 28 days of follow-up. The trial showed a statistically significant treatment effect favoring neratinib with a stratified HR of 0.66 [95% confidence interval (CI), 0.49-0.90, P = 0.008]. The estimated iDFS rate at 2 years was 94.2% (95% CI, 92.6%-95.4%) in patients treated with neratinib versus 91.9% (95% CI, 90.2%-93.2%) in those receiving placebo. Diarrhea was the most common adverse event (AE), with a 40% incidence of grade 3 or 4 diarrhea, and represents the most common AE leading to treatment discontinuation. Other frequent AEs (>10% incidence) were nausea, abdominal pain, fatigue, vomiting, rash, stomatitis, decreased appetite, and muscle spasms. Other than diarrhea, neratinib is associated with a low incidence of severe AEs; toxicities are generally reversible and manageable with dose interruptions, dose reductions, and/or standard medical care. This article summarizes FDA decision-making and data supporting the neratinib approval. Clin Cancer Res; 24(15); 3486-91. ©2018 AACRSee related commentary by Unni et al., p. 3483.

FDA Approval: Ribociclib for the Treatment of Postmenopausal Women with Hormone Receptor-Positive, HER2-Negative Advanced or Metastatic Breast Cancer.

On March 13, 2017, the FDA approved ribociclib (KISQALI; Novartis Pharmaceuticals Corp.), a cyclin-dependent kinase 4/6 inhibitor, in combination with an aromatase inhibitor as initial endocrine-based therapy for the treatment of postmenopausal women with hormone receptor (HR)-positive, HER2-negative advanced or metastatic breast cancer. The approval was based on a randomized, double-blind, placebo-controlled, international clinical trial (MONALEESA-2). A total of 668 patients were randomized to receive either ribociclib plus letrozole (n = 334) or placebo plus letrozole (n = 334). An improvement in progression-free survival (PFS) was observed in patients receiving ribociclib plus letrozole compared with patients receiving placebo plus letrozole [HR = 0.556; 95% confidence interval (CI), 0.429-0.720]. Overall response rate (ORR) in patients with measurable disease was 52.7% (95% CI, 46.6-58.9) in the ribociclib plus letrozole arm and 37.1% (95% CI, 31.1-43.2) in the placebo plus letrozole arm. Overall survival data were immature. The most common adverse reactions observed in 20% or more of patients taking ribociclib were neutropenia, nausea, fatigue, diarrhea, leukopenia, alopecia, vomiting, constipation, headache, and back pain. This article summarizes FDA decision-making and data supporting the approval of ribociclib. Clin Cancer Res; 24(13); 2999-3004. ©2018 AACRSee related commentary by Spring and Bardia, p. 2981.

FDA Approval Summary: Niraparib for the Maintenance Treatment of Patients with Recurrent Ovarian Cancer in Response to Platinum-Based Chemotherapy.

The FDA approved niraparib, a poly(ADP-ribose) polymerase (PARP) inhibitor, on March 27, 2017, for maintenance treatment of patients with recurrent epithelial ovarian, fallopian tube, or primary peritoneal cancer who are in response to platinum-based chemotherapy. Approval was based on data from the NOVA trial comparing niraparib with placebo in two independent cohorts, based on germline BRCA mutation status (gBRCAm vs. non-gBRCAm). Progression-free survival (PFS) in each cohort was the primary endpoint. In the gBRCAm cohort, estimated median PFS on niraparib was 21 months versus 5.5 months on placebo [HR, 0.26; 95% confidence interval (CI), 0.17-0.41; P < 0.0001]. In the non-gBRCAm cohort, estimated median PFS for niraparib and placebo was 9.3 and 3.9 months, respectively (HR, 0.45; 95% CI, 0.34-0.61; P < 0.0001). Common adverse reactions (>20% and higher incidence in the niraparib arm) with niraparib included thrombocytopenia, anemia, neutropenia, nausea, constipation, vomiting, mucositis, fatigue, decreased appetite, headache, insomnia, nasopharyngitis, dyspnea, rash, and hypertension. There were five cases of myelodysplastic syndrome and acute myeloid leukemia (1.4%) in patients treated with niraparib compared with two cases (1.1%) on placebo. Niraparib is the first PARP inhibitor approved as maintenance therapy for patients with ovarian, fallopian tube, or primary peritoneal cancer, with improvement in PFS, regardless of gBRCAm status. Clin Cancer Res; 24(17); 4066-71. ©2018 AACRSee related commentary by Konstantinopoulos and Matulonis, p. 4062.

FDA Approval Summary: Rucaparib for the Treatment of Patients with Deleterious BRCA Mutation-Associated Advanced Ovarian Cancer.

On December 19, 2016, the FDA granted accelerated approval to rucaparib (RUBRACA; Clovis Oncology, Inc.) for the treatment of patients with deleterious BRCA mutation (germline and/or somatic)-associated advanced ovarian cancer who have been treated with two or more chemotherapies. The FDA also approved the FoundationFocus CDx BRCA test (Foundation Medicine, Inc.), the first next-generation sequencing-based companion diagnostic, for identifying patients with advanced ovarian cancer eligible for treatment with rucaparib based on detection of deleterious BRCA1 and/or BRCA2 mutations in tumor tissue. Rucaparib's approval was based primarily on efficacy data from 106 patients with BRCA mutation-associated ovarian cancer who had prior treatment with two or more chemotherapies and safety data from 377 patients with ovarian cancer treated with rucaparib 600 mg orally twice daily on two open-label, single-arm trials. Investigator-assessed objective response rate was 54% [57/106; 95% confidence interval (CI), 44-64], and median duration of response was 9.2 months (95% CI, 6.6-11.7). The approved companion diagnostic verified tumor BRCA mutation status retrospectively in 96% (64/67) of patients. Common adverse reactions (≥20%) to rucaparib were nausea, fatigue, vomiting, anemia, abdominal pain, dysgeusia, constipation, decreased appetite, diarrhea, thrombocytopenia, and dyspnea. This article summarizes the FDA review and data supporting rucaparib's accelerated approval. Clin Cancer Res; 23(23); 7165-70. ©2017 AACRSee related commentary by Kohn et al., p. 7155.

U.S. Food and Drug Administration Approval: Cabozantinib for the Treatment of Advanced Renal Cell Carcinoma.

On April 25, 2016, the FDA approved cabozantinib (Cabometyx; Exelixis, Inc.) for the treatment of advanced renal cell carcinoma (RCC) in patients who have received prior antiangiogenic therapy. The approval was based on data from one randomized, open-label, multicenter study in which patients with RCC who had received prior antiangiogenic therapy were treated with either cabozantinib 60 mg orally once daily (n = 330) or everolimus 10 mg orally once daily (n = 328). The major efficacy outcome measure was progression-free survival (PFS) as assessed by a blinded independent radiology review committee in the first 375 randomized patients. A statistically significant improvement in PFS was seen, with a median PFS of 7.4 and 3.8 months in the cabozantinib and everolimus arms, respectively [hazard ratio (HR), 0.58; 95% confidence interval (CI), 0.45-0.74; P < 0.0001]. At a second interim analysis, a statistically significant improvement in overall survival (OS) in the intent-to-treat population was also demonstrated, with a median OS of 21.4 and 16.5 months in the cabozantinib and everolimus arms, respectively (HR, 0.66; 95% CI, 0.53-0.83; P = 0.0003). The most common (greater than or equal to 25%) adverse reactions included diarrhea, fatigue, nausea, decreased appetite, palmar-plantar erythrodysesthesia syndrome, hypertension, vomiting, weight loss, and constipation. Clin Cancer Res; 23(2); 330-5. ©2016 AACR.

Nonclinical Evaluations of Small-Molecule Oncology Drugs: Integration into Clinical Dose Optimization and Toxicity Management.

Multidisciplinary approaches that incorporate nonclinical pharmacologic and toxicologic characterization of small-molecule oncology drugs into clinical development programs may facilitate improved benefit-risk profiles and clinical toxicity management in patients. The performance of the current nonclinical safety-testing scheme was discussed, highlighting current strengths and areas for improvement. While current nonclinical testing appears to predict the clinical outcome where the prevalence of specific adverse effects are high, nonclinical testing becomes less reliable for predicting clinical adverse effects that occur infrequently, as with some kinase inhibitors. Although adverse effects associated with kinase inhibitors can often be predicted on the basis of target biology, drugs can be promiscuous and inhibit targets with poorly defined function and associated risks. Improvements in adverse effect databases and better characterization of the biologic activities of drug targets may enable better use of computational modeling approaches in predicting adverse effects with kinase inhibitors. Assessing safety of a lead candidate in parallel with other drug properties enables incorporation of a molecule's best features during chemical design, eliminates the worst molecules early, and permits timely investigation/characterization of toxicity mechanisms for identified liabilities. A safety lead optimization and candidate identification strategy that reduces intrinsic toxicity and metabolic risk and enhances selectivity can deliver selective kinase inhibitors that demonstrate on-target adverse effects identified nonclinically. Integrating clinical and nonclinical data during drug development can facilitate better identification and management of oncology drugs. Follow-up nonclinical studies may be used to better understand the risks in a given patient population and minimize or manage these risks more appropriately. Clin Cancer Res; 22(11); 2618-22. ©2016 AACR SEE ALL ARTICLES IN THIS CCR FOCUS SECTION, "NEW APPROACHES FOR OPTIMIZING DOSING OF ANTICANCER AGENTS".

FDA Approval of Palbociclib in Combination with Fulvestrant for the Treatment of Hormone Receptor-Positive, HER2-Negative Metastatic Breast Cancer.

On February 19, 2016, the FDA approved palbociclib (Ibrance, Pfizer) for use in combination with fulvestrant (Faslodex, AstraZeneca) for the treatment of women with hormone receptor (HR)-positive, HER2-negative advanced or metastatic breast cancer (MBC) with disease progression following endocrine therapy. The approval was based on the results of a randomized, double-blind, placebo-controlled trial conducted in 521 pre- and postmenopausal women with HR-positive, HER2-negative advanced or MBC. Patients were randomized (2:1) to receive palbociclib plus fulvestrant (n = 347) or placebo plus fulvestrant (n = 174). The primary endpoint was investigator-assessed progression-free survival (PFS). A statistically significant and clinically meaningful improvement in PFS (9.5 months vs. 4.6 months) was observed in patients receiving palbociclib plus fulvestrant [HR 0.46; 95% confidence interval (CI), 0.36-0.59; P < 0.0001]. Safety data confirmed the known adverse reaction profile of palbociclib. The most common adverse reactions (>20%) in patients treated with palbociclib were neutropenia, leukopenia, infections, fatigue, nausea, anemia, stomatitis, headache, diarrhea, and thrombocytopenia. This approval was granted in the context of a prior accelerated approval for palbociclib in combination with letrozole in patients with HR-positive, HER2-negative advanced breast cancer as initial endocrine-based therapy. Clin Cancer Res; 22(20); 4968-72. ©2016 AACR.

FDA Approval: Uridine Triacetate for the Treatment of Patients Following Fluorouracil or Capecitabine Overdose or Exhibiting Early-Onset Severe Toxicities Following Administration of These Drugs.

On December 11, 2015, the FDA approved uridine triacetate (VISTOGARD; Wellstat Therapeutics Corporation) for the emergency treatment of adult and pediatric patients following a fluorouracil or capecitabine overdose regardless of the presence of symptoms, and of those who exhibit early-onset, severe, or life-threatening toxicity affecting the cardiac or central nervous system, and/or early onset, unusually severe adverse reactions (e.g., gastrointestinal toxicity and/or neutropenia) within 96 hours following the end of fluorouracil or capecitabine administration. Uridine triacetate is not recommended for the nonemergent treatment of adverse reactions associated with fluorouracil or capecitabine because it may diminish the efficacy of these drugs, and the safety and efficacy of uridine triacetate initiated more than 96 hours following the end of administration of these drugs has not been established. The approval is based on data from two single-arm, open-label, expanded-access trials in 135 patients receiving uridine triacetate (10 g or 6.2 g/m(2) orally every 6 hours for 20 doses) for fluorouracil or capecitabine overdose, or who exhibited severe or life-threatening toxicities within 96 hours following the end of fluorouracil or capecitabine administration. Ninety-six percent of patients met the major efficacy outcome measure, which was survival at 30 days or survival until the resumption of chemotherapy, if prior to 30 days. The most common adverse reactions were vomiting, nausea, and diarrhea. This article summarizes the FDA review of this New Drug Application, the data supporting approval of uridine triacetate, and the unique regulatory situations encountered by this approval. Clin Cancer Res; 22(18); 4545-49. ©2016 AACR.

Rac1b, a tumor associated, constitutively active Rac1 splice variant, promotes cellular transformation.

A novel splice variant of Rac1, designated Rac1b, is expressed in human breast and colon carcinoma cells. Rac1b contains an additional 19 amino-acid insert immediately behind the switch II domain, a region important for Rac1 interaction with regulators and effectors. Recent studies showed that Rac1b exhibited the biochemical properties of a constitutively activated GTPase, yet it showed impaired interaction with downstream effectors, suggesting that Rac1b may be defective in biological activity. Whether Rac1b is a biologically active protein was not addressed. Therefore, we evaluated the biochemical, signaling and growth-promoting properties of authentic Rac1b. Similar to previous observations, we found that Rac1b showed enhanced intrinsic guanine nucleotide exchange activity, impaired intrinsic GTPase activity, and failed to interact with RhoGDI. Surprisingly, we found that Rac1b, like the constitutively-activated and transforming Rac1(Q61L) mutant, promoted growth transformation of NIH3T3 cells. Rac1b-expressing cells also showed a loss of density-dependent and anchorage-dependent growth. Surprisingly, unlike activated Rac1(61L), Rac1b did not show enhanced activation of the nuclear factor kappaB (NF-kappaB) transcription factor or stimulate cyclin D1 expression, the signaling activities that best correlate with Rac1 transforming activity. However, Rac1b did promote activation of the AKT serine/threonine kinase. Therefore, we suggest that Rac1b selectively activates a subset of Rac1 downstream signaling pathways to facilitate cellular transformation.

Vav transformation requires activation of multiple GTPases and regulation of gene expression.

Although Vav can act as a guanine nucleotide exchange factor for RhoA, Rac1, and Cdc42, its transforming activity has been ascribed primarily to its ability to activate Rac1. However, because activated Vav, but not Rac-specific guanine nucleotide exchange factors, exhibits very potent focus-forming transforming activity when assayed in NIH 3T3 cells, Vav transforming activity must also involve activation of Rac-independent pathways. In this study, we determined the involvement of other Rho family proteins and their signaling pathways in Vav transformation. We found that RhoA, Rac1, and Cdc42 functions are all required for Vav transforming activity. Furthermore, we determined that Vav activation of nuclear factor-kappaB and the Jun NH2-terminal kinase mitogen-activated protein kinase (MAPK) is necessary for full transformation by Vav, whereas p38 MAPK does not seem to play an important role. We also determined that Vav is a weak activator of Elk-1 via a Ras- and MAPK/extracellular signal-regulated kinase kinase-dependent pathway, and this activity was essential for Vav transformation. Thus, we conclude that full Vav transforming activation is mediated by the activation of multiple small GTPases and their subsequent activation of signaling pathways that regulate changes in gene expression. Because Vav is activated by the epidermal growth factor receptor and other tyrosine kinases involved in cancer development, defining the role of aberrant Vav signaling may identify activities of receptor tyrosine kinases important for human oncogenesis.

U.s. Food and Drug Administration approval: carfilzomib for the treatment of multiple myeloma.

The U.S. Food and Drug Administration (FDA) review leading to accelerated approval of carfilzomib is described. A single-arm trial enrolled 266 patients with multiple myeloma refractory to the most recent therapy who had received prior treatment with bortezomib and an immunomodulatory agent (IMID). Patients received carfilzomib by intravenous infusion over 2 to 10 minutes at a dose of 20 mg/m2 on days 1, 2, 8, 9, 15, and 16 of the 28 days of cycle 1, and at a dose of 27 mg/m2 on the same schedule in cycle 2 and subsequent cycles. The primary efficacy endpoint was overall response rate (ORR) as determined by an independent review committee using International Myeloma Working Group Uniform Response Criteria. The safety of carfilzomib was evaluated in 526 patients with multiple myeloma treated with various dosing regimens. The ORR was 23%. The median duration of response was 7.8 months. The most common adverse reactions associated with carfilzomib infusion were fatigue, anemia, nausea, thrombocytopenia, dyspnea, diarrhea, and fever. The most common serious adverse events were pneumonia, acute renal failure, fever, and congestive heart failure. Infusion reactions to carfilzomib could be reduced by pretreatment with dexamethasone and intravenous fluids. On July 20, 2012, the FDA granted accelerated approval of carfilzomib for the treatment of patients with multiple myeloma who have received at least two prior therapies including bortezomib and an IMID and who have shown disease progression while on therapy or within 60 days of completion of the last therapy.

Semaphorin 3E initiates antiangiogenic signaling through plexin D1 by regulating Arf6 and R-Ras.

Recent studies revealed that a class III semaphorin, semaphorin 3E (Sema3E), acts through a single-pass transmembrane receptor, plexin D1, to provide a repulsive cue for plexin D1-expressing endothelial cells, thus providing a highly conserved and developmentally regulated signaling system guiding the growth of blood vessels. We show here that Sema3E acts as a potent inhibitor of adult and tumor-induced angiogenesis. Activation of plexin D1 by Sema3E causes the rapid disassembly of integrin-mediated adhesive structures, thereby inhibiting endothelial cell adhesion to the extracellular matrix (ECM) and causing the retraction of filopodia in endothelial tip cells. Sema3E acts on plexin D1 to initiate a two-pronged mechanism involving R-Ras inactivation and Arf6 stimulation, which affect the status of activation of integrins and their intracellular trafficking, respectively. Ultimately, our present study provides a molecular framework for antiangiogenesis signaling, thus impinging on a myriad of pathological conditions that are characterized by aberrant increase in neovessel formation, including cancer.

Critical role of the pleckstrin homology and cysteine-rich domains in Vav signaling and transforming activity.

Vav family proteins are members of the Dbl family of guanine nucleotide exchange factors and activators of Rho family small GTPases. In addition to the Dbl homology (DH) domain important for guanine nucleotide exchange factor catalytic function, all Dbl family proteins contain an adjacent pleckstrin homology (PH) domain that serves to regulate DH domain activity. Although the role of the PH domain in Vav function has been evaluated extensively, its precise role and whether it serves a distinct role in different Vav proteins remain unresolved. Additionally, the precise role of an adjacent cysteine-rich domain (CRD) in regulating DH domain function is also unclear. In this study, we evaluated the contribution of these putative protein-protein or protein-lipid interaction domains to Vav signaling and transforming activity. In contrast to previous observations, we found that the PH domain is critical for Vav transforming activity. Similarly, the CRD was also essential and served a function distinct from that of the PH domain. Although mutation of either domain reduced Vav membrane association, addition of plasma membrane targeting sequences to either the CRD or PH domain mutant proteins did not restore Vav transforming activity. This result contrasts with other Dbl family proteins, where a membrane targeting sequence alone was sufficient to restore the loss of function caused by mutation of the PH domain. Furthermore, green fluorescent protein fusion proteins containing the PH domain or CRD, or both, failed to target to the plasma membrane, suggesting that these two domains also serve regulatory functions independent of promoting membrane localization. Finally, we found that phosphatidylinositol 3-kinase activation may promote Vav membrane association via phosphatidylinositol 3,4,5-triphosphate binding to the PH domain.