De-escalation of axillary surgery in patients outside Z0011 criteria.

Over the past 2 decades, axillary surgical management for breast cancer patients has been reshaped after several practice-changing randomized clinical trials provided evidence to support the de-escalation of axillary surgery, specifically the omission of axillary lymph node dissection, for patients with positive axillary lymph nodes. One such practice-changing trial was the American College of Surgeons Oncology Group Z0011 trial, which showed that patients with clinical T1-2 breast tumors and limited nodal disease (1-2 positive sentinel lymph nodes) who underwent upfront breast-conserving therapy could be safely spared the morbidity of axillary lymph node dissection. American College of Surgeons Oncology Group Z0011 has been criticized as several important groups were excluded, such as patients who underwent a mastectomy, patients with >2 positive sentinel lymph nodes, or patients with imaging-detected lymph node metastases. These exclusions have led to unclear guidelines and very difficult management decisions for many patients with breast cancer who are just outside the Z0011 criteria. Several subsequent trials that investigated sentinel lymph node biopsy alone or sentinel lymph node biopsy plus axillary radiation versus axillary lymph node dissection enrolled patients with higher volumes of disease than American College of Surgeons Oncology Group Z0011, such as mastectomy patients or patients with >2 positive sentinel lymph nodes. The goal of this review is to describe the findings of these trials and to discuss the current best practices regarding axillary management in patients who are candidates for upfront surgery but were excluded from American College of Surgeons Oncology Group Z0011, with a particular focus on patients undergoing mastectomy, patients with >2 positive sentinel lymph nodes, patients with large or multifocal tumors, and patients with imaging-detected biopsy-proven lymph node metastases.

Preoperative MRI and Its Impact on Surgical Outcomes in Patients with Triple Negative Breast Cancer Treated with Primary Surgery: Did New Margin Guidelines or Cavity Shave Margins Practice Diminish the Role of Preoperative MRI?

BACKGROUND: Results of an earlier retrospective study from our institution suggested that patients with triple negative breast cancer (TNBC) who had preoperative MRI may have had an improved local recurrence rate (LRR) after breast conserving surgery (BCS). We aimed to clarify the impact of preoperative MRI on surgical outcomes in an expanded TNBC cohort treated by BCS in a contemporary era. METHODS: Our study cohort comprised 648 patients with TNBC who underwent BCS between 2009 and 2018. Demographic and clinical characteristics were compared between those with (n = 292, 45.1%) and without (n = 356, 54.9%) preoperative MRI. Multivariable logistic regression was performed to assess the association of preoperative MRI with surgical outcomes. RESULTS: The crude LRR of 3.5% was lower than previously reported. Univariable analyses demonstrated that the LRR and re-excision rates in the MRI and no-MRI groups were 3.4 and 3.7%, 21.6% and 27.2%, p = 0.876 and p = 0.10, respectively. Multivariable logistic regression analyses demonstrated that preoperative MRI was not associated with a lower LRR: odds ratio (OR) = 1.42 (p = 0.5). During our study period, new margin guidelines and shave margins practice were adopted in 2014 and 2015. To account for their effects, the year of diagnosis/surgery and other clinical variables were adjusted in multivariable logistic regression and inverse probability weighting models to demonstrate that preoperative MRI remained associated with a lower re-excision risk, OR 0.56, p = 0.04l; and a lower re-excision rate, 23.15% versus 36.0%, p < 0.01, respectively. CONCLUSIONS: Our findings suggested that patients with TNBC anticipating BCS may benefit from preoperative MRI.

NOTCH3-targeted antibody drug conjugates regress tumors by inducing apoptosis in receptor cells and through transendocytosis into ligand cells.

Aberrant NOTCH3 signaling and overexpression is oncogenic, associated with cancer stem cells and drug resistance, yet therapeutic targeting remains elusive. Here, we develop NOTCH3-targeted antibody drug conjugates (NOTCH3-ADCs) by bioconjugation of an auristatin microtubule inhibitor through a protease cleavable linker to two antibodies with differential abilities to inhibit signaling. The signaling inhibitory antibody rapidly induces ligand-independent receptor clustering and internalization through both caveolin and clathrin-mediated pathways. The non-inhibitory antibody also efficiently endocytoses via clathrin without inducing receptor clustering but with slower lysosomal co-localization kinetics. In addition, DLL4 ligand binding to the NOTCH3 receptor mediates transendocytosis of NOTCH3-ADCs into ligand-expressing cells. NOTCH3-ADCs internalize into receptor and ligand cells independent of signaling and induce cell death in both cell types representing an atypical mechanism of ADC cytotoxicity. Treatment of xenografts with NOTCH3-ADCs leads to sustained tumor regressions, outperforms standard-of-care chemotherapy, and allows targeting of tumors that overexpress NOTCH3 independent of signaling inhibition.

Development of Highly Optimized Antibody-Drug Conjugates against CD33 and CD123 for Acute Myeloid Leukemia.

PURPOSE: Mortality due to acute myeloid leukemia (AML) remains high, and the management of relapsed or refractory AML continues to be therapeutically challenging. The reapproval of Mylotarg, an anti-CD33-calicheamicin antibody-drug conjugate (ADC), has provided a proof of concept for an ADC-based therapeutic for AML. Several other ADCs have since entered clinical development of AML, but have met with limited success. We sought to develop a next-generation ADC for AML with a wide therapeutic index (TI) that overcomes the shortcomings of previous generations of ADCs. EXPERIMENTAL DESIGN: We compared the TI of our novel CD33-targeted ADC platform with other currently available CD33-targeted ADCs in preclinical models of AML. Next, using this next-generation ADC platform, we performed a head-to-head comparison of two attractive AML antigens, CD33 and CD123. RESULTS: Our novel ADC platform offered improved safety and TI when compared with certain currently available ADC platforms in preclinical models of AML. Differentiation between the CD33- and CD123-targeted ADCs was observed in safety studies conducted in cynomolgus monkeys. The CD33-targeted ADC produced severe hematologic toxicity, whereas minimal hematologic toxicity was observed with the CD123-targeted ADC at the same doses and exposures. The improved toxicity profile of an ADC targeting CD123 over CD33 was consistent with the more restricted expression of CD123 in normal tissues. CONCLUSIONS: We optimized all components of ADC design (i.e., leukemia antigen, antibody, and linker-payload) to develop an ADC that has the potential to translate into an effective new therapy against AML.

Kisspeptin deficiency leads to abnormal adrenal glands and excess steroid hormone secretion.

Knockout mice for the kisspeptin receptor, Kiss1r (Kiss1r-/-) and its ligand kisspeptin, Kiss1 (Kiss1-/-) replicate the phenotype of isolated hypogonadotropic hypogonadism (IHH) associated with variants of these genes in humans. A recent report suggests that kisspeptin may be involved in human fetal adrenocortical development and function. Herein, we characterized the adrenal function and morphology in Kiss1-/- mice that do not go through normal puberty. Two fetal markers were expressed in eosinophilic cells potentially derived from the X-zone that should disappear at puberty in male mice and during the first pregnancy in female animals. Although the hypercorticosteronism observed in Kiss1-/- females corrected overtime, hyperaldosteronism persisted at 14 months and correlated with the overexpression of Star. To determine if KISS1 and KISS1R genes are involved in the development of primary aldosteronism (PA) and hypercortisolism [Cushing's syndrome (CS)] in humans, we sequenced these 2 genes in 65 patients with PA and/or CS. Interestingly, a patient with CS presented with a germline KISS1 variant (p.H90D, rs201073751). We also found three rare variants in the KISS1R gene in three patients with PA: p.C95W (rs141767649), p.A189T (rs73507527) and p.R229R (rs115335009). The two missense variants have been previously associated with IHH. Our findings suggest that KISS1 may play a role in adrenal function in mice and possibly adrenocortical steroid hormone secretion in humans, beyond its recently described role in human fetal adrenocortical development.

Multidimensional Coculture System to Model Lung Squamous Carcinoma Progression.

Tumor-stroma interactions play a critical role in the development of lung squamous carcinoma (LUSC). However, understanding how these dynamic interactions contribute to tissue architectural changes observed during tumorigenesis remains challenging due to the lack of appropriate models. In this protocol, we describe the generation of a 3D coculture model using a LUSC primary cell culture known as TUM622. TUM622 cells were established from a LUSC patient-derived xenograft (PDX) and have the unique property to form acinar-like structures when seeded in a basement membrane matrix. We demonstrate that TUM622 acini in 3D coculture recapitulate key features of tissue architecture during LUSC progression as well as the dynamic interactions between LUSC cells and components of the tumor microenvironment (TME), including the extracellular matrix (ECM) and cancer-associated fibroblasts (CAFs). We further adapt our principal 3D culturing protocol to demonstrate how this system could be utilized for various downstream analyses. Overall, this organoid model creates a biologically rich and adaptable platform that enables one to gain insight into the cell-intrinsic and extrinsic mechanisms that promote the disruption of epithelial architectures during carcinoma progression and will aid the search for new therapeutic targets and diagnostic markers.

Pregnancy Loss and Iodine Status: The LIFE Prospective Cohort Study.

Iodine deficiency in pregnancy is a common problem in the United States and parts of Europe, but whether iodine deficiency is associated with increased pregnancy loss has not been well studied. The LIFE study provided an excellent opportunity to examine the relationship between iodine status and pregnancy loss because women were monitored prospectively to ensure excellent ascertainment of conceptions. The LIFE study, a population-based prospective cohort study, monitored 501 women who had discontinued contraception within two months to become pregnant; 329 became pregnant, had urinary iodine concentrations measured on samples collected at enrollment, and were followed up to determine pregnancy outcomes. Of the 329, 196 had live births (59.5%), 92 (28.0%) had losses, and 41 (12.5%) withdrew or were lost to follow up. Urinary iodine concentrations were in the deficiency range in 59.6% of the participants. The risk of loss, however, was not elevated in the mildly deficient group (hazard ratio 0.69, 95% confidence interval 0.34, 1.38), the moderately deficient group (hazard ratio 0.81, 95% confidence interval 0.43, 1.51), or the severely deficient group (hazard ratio 0.69, 95% confidence interval 0.32, 1.50). Iodine deficiency, even when moderate to severe, was not associated with increased rates of pregnancy loss. This study provides some reassurance that iodine deficiency at levels seen in many developed countries does not increase the risk of pregnancy loss.

Do patients with familial nonmedullary thyroid cancer present with more aggressive disease? Implications for initial surgical treatment.

BACKGROUND: There are conflicting reports on whether familial nonmedullary thyroid cancer is more aggressive than sporadic nonmedullary thyroid cancer. Our aim was to determine if the clinical and pathologic characteristics of familial nonmedullary thyroid cancer are different than nonmedullary thyroid cancer. METHODS: We compared patients with familial nonmedullary thyroid cancer to a cohort of 53,571 nonmedullary thyroid cancer patients from the Surveillance, Epidemiology, and End Results database. RESULTS: A total of 78 patients with familial nonmedullary thyroid cancer from 31 kindreds presented at a younger age (P = .04) and had a greater rate of T1 disease (P = .019), lymph node metastasis (P = .002), and the classic variant of papillary thyroid cancer on histology (P < .001) compared with the Surveillance, Epidemiology, and End Results cohort. Patients with ≥3 affected family members presented at a younger age (P = .04), had a lesser female-to-male ratio (P = .04), and had a greater rate of lymph node metastasis (P = .009). Compared with the Surveillance, Epidemiology, and End Results cohort, we found a higher prevalence of lymph node metastasis in familial nonmedullary thyroid cancer index cases (P = .003) but not in those diagnosed by screening ultrasonography (P = .58). CONCLUSION: Patients with familial nonmedullary thyroid cancer present at a younger age and have a greater rate of lymph node metastasis. The treatment for familial nonmedullary thyroid cancer should be more aggressive in patients who present clinically and in those who have ≥3 first-degree relatives affected.

Cancer-associated fibroblasts suppress SOX2-induced dysplasia in a lung squamous cancer coculture.

Tumorigenesis depends on intricate interactions between genetically altered tumor cells and their surrounding microenvironment. While oncogenic drivers in lung squamous carcinoma (LUSC) have been described, the role of stroma in modulating tissue architecture, particularly cell polarity, remains unclear. Here, we report the establishment of a 3D coculture system of LUSC epithelial cells with cancer-associated fibroblasts (CAFs) and extracellular matrix that together capture key components of the tumor microenvironment (TME). Single LUSC epithelial cells develop into acinar-like structures with 0.02% efficiency, and addition of CAFs provides proper tumor-stromal interactions within an appropriate 3D architectural context. Using this model, we recapitulate key pathological changes during tumorigenesis, from hyperplasia to dysplasia and eventually invasion, in malignant LUSC spheroids that undergo phenotypic switching in response to cell intrinsic and extrinsic changes. Overexpression of SOX2 is sufficient to mediate the transition from hyperplasia to dysplasia in LUSC spheroids, while the presence of CAFs makes them invasive. Unexpectedly, CAFs suppress the activity of high SOX2 levels, restore hyperplasia, and enhance the formation of acinar-like structures. Taken together, these observations suggest that stromal factors can override cell intrinsic oncogenic changes in determining the disease phenotype, thus providing fundamental evidence for the existence of dynamic reciprocity between the nucleus and the TME of LUSC.

Copy number variants in hypoplastic right heart syndrome.

Hypoplastic right heart syndrome (HRHS) is a rare congenital defect characterized by underdeveloped and malformed structures of the right heart. Familial recurrence of HRHS indicates genetic factors contribute to its etiology. Our study investigates the presence of copy number variants (CNVs) in HRHS cases. We genotyped 42 HRHS cases identified from live births throughout California (2003-2010) using the Illumina HumanOmni2.5-8 array. We identified 14 candidate CNVs in 14 HRHS cases (33%) based on the genes included in the CNVs and their functions. Duplications overlapping part of ERBB4 were identified in two unrelated cases. ERBB4 is a neuregulin receptor with a pivotal role in cardiomyocyte differentiation and heart development. We also described a 7.5 Mb duplication at 16q11-12. Multiple genes in the duplicated region have previously been linked to heart defects and cardiac development, including RPGRIP1L, RBL2, SALL1, and MYLK3. Of the 14 validated CNVs, we identified four CNVs in close proximity to genes linked to the Wnt signaling pathway. This study expands on our previous work supporting the role of genetics in HRHS. We identified CNVs affecting crucial genes and signaling pathways involved in right heart development. ERBB4 and duplication of the 16q11-12 region are important areas for future investigation.

Rare copy number variants identified in prune belly syndrome.

Prune belly syndrome (PBS), also known as Eagle-Barrett syndrome, is a rare congenital disorder characterized by absence or hypoplasia of the abdominal wall musculature, urinary tract anomalies, and cryptorchidism in males. The etiology of PBS is largely unresolved, but genetic factors are implicated given its recurrence in families. We examined cases of PBS to identify novel pathogenic copy number variants (CNVs). A total of 34 cases (30 males and 4 females) with PBS identified from all live births in New York State (1998-2005) were genotyped using Illumina HumanOmni2.5 microarrays. CNVs were prioritized if they were absent from in-house controls, encompassed ≥10 consecutive probes, were ≥20 Kb in size, had ≤20% overlap with common variants in population reference controls, and had ≤20% overlap with any variant previously detected in other birth defect phenotypes screened in our laboratory. We identified 17 candidate autosomal CNVs; 10 cases each had one CNV and four cases each had two CNVs. The CNVs included a 158 Kb duplication at 4q22 that overlaps the BMPR1B gene; duplications of different sizes carried by two cases in the intron of STIM1 gene; a 67 Kb duplication 202 Kb downstream of the NOG gene, and a 1.34 Mb deletion including the MYOCD gene. The identified rare CNVs spanned genes involved in mesodermal, muscle, and urinary tract development and differentiation, which might help in elucidating the genetic contribution to PBS. We did not have parental DNA and cannot identify whether these CNVs were de novo or inherited. Further research on these CNVs, particularly BMP signaling is warranted to elucidate the pathogenesis of PBS.

Copy number variants in Ebstein anomaly.

BACKGROUND: Ebstein anomaly (EA) is a rare congenital defect characterized by apical displacement of the septal tricuspid leaflets and atrialization of the right ventricle. The etiology of EA is unclear; however, recurrence in families and the association of EA with genetic syndromes and copy number variants (CNVs) suggest a genetic component. OBJECTIVE: We performed a population-based study to search for recurrent and novel CNVs in a previously unreported set of EA cases. METHODS: We genotyped 60 EA cases identified from all live births (2,891,076) from selected California counties (1991-2010) using the Illumina HumanOmni2.5-8 array. We identified 38 candidate CNVs in 28 (46%) cases and prioritized and validated 11 CNVs based on the genes included. RESULTS: Five CNVs (41%) overlapped or were close to genes involved in early myocardial development, including NODAL, PDLIM5, SIX1, ASF1A and FGF12. We also replicated a previous association of EA with CNVs at 1p34.1 and AKAP12. Finally, we identified four CNVs overlapping or in close proximity to the transcription factors HES3, TRIM71, CUX1 and EIF4EBP2. CONCLUSIONS: This study supports the relationship of genetic factors to EA and demonstrates that defects in cardiomyocytes and myocardium differentiation may play a role. Abnormal differentiation of cardiomyocytes and how genetic factors contribute should be examined for their association with EA.

The E3 ubiquitin ligase Siah1 regulates adrenal gland organization and aldosterone secretion.

Primary and secondary hypertension are major risk factors for cardiovascular disease, the leading cause of death worldwide. Elevated secretion of aldosterone resulting from primary aldosteronism (PA) is a key driver of secondary hypertension. Here, we report an unexpected role for the ubiquitin ligase Siah1 in adrenal gland development and PA. Siah1a-/- mice exhibit altered adrenal gland morphology, as reflected by a diminished X-zone, enlarged medulla, and dysregulated zonation of the glomerulosa as well as increased aldosterone levels and aldosterone target gene expression and reduced plasma potassium levels. Genes involved in catecholamine biosynthesis and cAMP signaling are upregulated in the adrenal glands of Siah1a-/- mice, while genes related to retinoic acid signaling and cholesterol biosynthesis are downregulated. Loss of Siah1 leads to increased expression of the Siah1 substrate PIAS1, an E3 SUMO protein ligase implicated in the suppression of LXR, a key regulator of cholesterol levels in the adrenal gland. In addition, SIAH1 sequence variants were identified in patients with PA; such variants impaired SIAH1 ubiquitin ligase activity, resulting in elevated PIAS1 expression. These data identify a role for the Siah1-PIAS1 axis in adrenal gland organization and function and point to possible therapeutic targets for hyperaldosteronism.

Purine Nucleotide Availability Regulates mTORC1 Activity through the Rheb GTPase.

Pharmacologic agents that interfere with nucleotide metabolism constitute an important class of anticancer agents. Recent studies have demonstrated that mTOR complex 1 (mTORC1) inhibitors suppress de novo biosynthesis of pyrimidine and purine nucleotides. Here, we demonstrate that mTORC1 itself is suppressed by drugs that reduce intracellular purine nucleotide pools. Cellular treatment with AG2037, an inhibitor of the purine biosynthetic enzyme GARFT, profoundly inhibits mTORC1 activity via a reduction in the level of GTP-bound Rheb, an obligate upstream activator of mTORC1, because of a reduction in intracellular guanine nucleotides. AG2037 treatment provokes both mTORC1 inhibition and robust tumor growth suppression in mice bearing non-small-cell lung cancer (NSCLC) xenografts. These results indicate that alterations in purine nucleotide availability affect mTORC1 activity and suggest that inhibition of mTORC1 contributes to the therapeutic effects of purine biosynthesis inhibitors.

The Elipse Balloon, a swallowable gastric balloon for weight loss not requiring sedation, anesthesia or endoscopy: a pilot study with 12-month outcomes.

BACKGROUND: The Elipse Balloon is a swallowable gastric balloon for weight loss that can be deployed without the use of endoscopy or anesthesia. This study aims to report on 12-month safety and efficacy outcomes. SETTING: Private hospital, Athens, Greece. METHODS: This was a prospective, nonrandomized open trial. Twelve patients between 18 and 64 years of age with a body mass index (BMI) of 27.0 to 40.0 kg/m2 were enrolled. The Elipse capsule is swallowed with water and its location in the stomach is confirmed by x-ray. All patients received diet and exercise counseling only during the therapy period (0-4 mo). Eight months later they were seen in clinic to assess 12-month outcomes. RESULTS: There were 7 (58.3%) females and 5 (41.7%) males. The mean age was 41 years, mean BMI was 35.9 kg/m2, mean weight was 103.5±15.8 kg, and mean waist circumference was 117.6±14.9 cm. All patients swallowed the balloon. Eleven (91.7%) balloons were filled and were included in subsequent analysis. All balloons were excreted safely, and no serious adverse events were reported. Accommodative symptoms were not severe and of very short duration. Mean excess weight loss percentage and total weight loss percentage were 50.2% and 14.6% at balloon excretion and 17.6% and 5.9% at 12 months, respectively. There was a statistically significant improvement in patients' weight, BMI, body fat, waist circumference, diastolic blood pressure, HbA1C, cholesterol, thyroid stimulating hormone, aspartate transaminase, and alanine transaminase at balloon excretion. Quality of life was significantly improved at excretion and 12 months. CONCLUSIONS: This study is the first to demonstrate 12-month efficacy and performance outcomes of the Elipse Balloon.

Elipse, the first procedureless gastric balloon for weight loss: a prospective, observational, open-label, multicenter study.

Background and study aims Conventional gastric balloons for weight loss require endoscopy for placement and removal. The Elipse device is swallowed, resides in the stomach for 4 months, and is then expelled. The objectives of this study were to assess the safety of Elipse and to measure its effects on weight loss, metabolic parameters, and quality of life. Methods Each participant swallowed one Elipse device, which was filled with 550 mL of filling fluid through a thin delivery catheter that was then removed. Weight was measured every 2 weeks, and metabolic parameters and quality of life were assessed at baseline and at trial exit. Results 34 patients, with a mean body mass index of 34.8 kg/m2, were enrolled. All 34 patients successfully swallowed the Elipse device. All adverse events were either self-limiting or resolved with medication. All balloons were safely excreted. At 4 months, the mean percent total body weight loss was 10 %. Mean waist circumference was reduced by 8.4 cm. Improvements were also seen in hemoglobin A1c, triglycerides, low density lipoprotein, and blood pressure. At trial exit, quality of life measures had improved across all domains. Conclusion These results demonstrate clinically significant weight loss with the Elipse, the first procedureless gastric balloon. The weight loss was similar to that seen in previous studies of endoscopically placed balloons. In addition, Elipse therapy led to improvements in waist circumference, several metabolic parameters, and overall quality of life.ClinicalTrials.gov identifier: NCT 02802007.

Prospective 30-Day Outcome Evaluation of a Fast-Track Protocol for 23-Hour Ambulatory Primary and Revisional Laparoscopic Roux-en-Y Gastric Bypass in 820 Consecutive Unselected Patients.

BACKGROUND: Results from validated national databases suggest that a 1-day length of stay (LOS) is not only unattainable in more than 20% of primary laparoscopic Roux-en-Y gastric bypass (LRYGB) operations, but it is also associated with an increased 30-day mortality risk. There are no published data regarding the feasibility and safety of 1-day LOS after revisional LRYGB. STUDY DESIGN: We reviewed 1 surgeon's experience after implementation of a 1-day fast-track protocol (FTP) in 784 primary and 36 revisional consecutive LRYGB patients. Patient demographics, comorbidities, preoperative laboratory values, diagnostic work-up, operative characteristics, LOS, 30-day outcomes, and patient satisfaction scores were prospectively collected in a deidentified registry. Predictors of longer hospitalization were assessed by univariate and multiple regression analysis. RESULTS: One-day LOS was accomplished in 71.9% primary and 58.3% revisional LRYGBs. Median LOS was 1 day for primary and revisional LRYGB and mean lengths of stay were 1.34 and 1.63 days, respectively. "Excellent" or "very good" satisfaction ratings were reported regarding the FTP education provided (95.3%), surgeon's accessibility (98.3%), and hospital care (86.5%). Only 0.34% of patients who had a 1-day LOS indicated that they would benefit from an additional in-hospital stay. In multivariate analysis, independent predictors of longer hospitalization included prolonged operative time, hemoglobin reduction, reoperation for primary LRYGB, and operative time for revisional LRYGB. CONCLUSIONS: A 1-day FTP can be successfully implemented in the majority of primary and revisional LRYGB patients regardless of preoperative patient characteristics without increasing the risk of 30-day morbidity, mortality, reoperations, readmissions, emergency department or outpatient fluid visits, or reducing patient satisfaction.

Loss of MIG6 Accelerates Initiation and Progression of Mutant Epidermal Growth Factor Receptor-Driven Lung Adenocarcinoma.

UNLABELLED: Somatic mutations in the EGFR kinase domain drive lung adenocarcinoma. We have previously identified MIG6, an inhibitor of ERBB signaling and a potential tumor suppressor, as a target for phosphorylation by mutant EGFRs. Here, we demonstrate that MIG6 is a tumor suppressor for the initiation and progression of mutant EGFR-driven lung adenocarcinoma in mouse models. Mutant EGFR-induced lung tumor formation was accelerated in Mig6-deficient mice, even with Mig6 haploinsufficiency. We demonstrate that constitutive phosphorylation of MIG6 at Y394/Y395 in EGFR-mutant human lung adenocarcinoma cell lines is associated with an increased interaction of MIG6 with mutant EGFR, which may stabilize EGFR protein. MIG6 also fails to promote mutant EGFR degradation. We propose a model whereby increased tyrosine phosphorylation of MIG6 decreases its capacity to inhibit mutant EGFR. Nonetheless, the residual inhibition is sufficient for MIG6 to delay mutant EGFR-driven tumor initiation and progression in mouse models. SIGNIFICANCE: This study demonstrates that MIG6 is a potent tumor suppressor for mutant EGFR-driven lung tumor initiation and progression in mice and provides a possible mechanism by which mutant EGFR can partially circumvent this tumor suppressor in human lung adenocarcinoma.

Epigenetic reprogramming by tumor-derived EZH2 gain-of-function mutations promotes aggressive 3D cell morphologies and enhances melanoma tumor growth.

In addition to genetic alterations, cancer cells are characterized by myriad epigenetic changes. EZH2 is a histone methyltransferase that is over-expressed and mutated in cancer. The EZH2 gain-of-function (GOF) mutations first identified in lymphomas have recently been reported in melanoma (~2%) but remain uncharacterized. We expressed multiple EZH2 GOF mutations in the A375 metastatic skin melanoma cell line and observed both increased H3K27me3 and dramatic changes in 3D culture morphology. In these cells, prominent morphological changes were accompanied by a decrease in cell contractility and an increase in collective cell migration. At the molecular level, we observed significant alteration of the axonal guidance pathway, a pathway intricately involved in the regulation of cell shape and motility. Furthermore, the aggressive 3D morphology of EZH2 GOF-expressing melanoma cells (both endogenous and ectopic) was attenuated by EZH2 catalytic inhibition. Finally, A375 cells expressing exogenous EZH2 GOF mutants formed larger tumors than control cells in mouse xenograft studies. This study not only demonstrates the first functional characterization of EZH2 GOF mutants in non-hematopoietic cells, but also provides a rationale for EZH2 catalytic inhibition in melanoma.

Long-term tumor regression induced by an antibody-drug conjugate that targets 5T4, an oncofetal antigen expressed on tumor-initiating cells.

Antibody-drug conjugates (ADC) represent a promising therapeutic modality for the clinical management of cancer. We sought to develop a novel ADC that targets 5T4, an oncofetal antigen expressed on tumor-initiating cells (TIC), which comprise the most aggressive cell population in the tumor. We optimized an anti-5T4 ADC (A1mcMMAF) by sulfydryl-based conjugation of the humanized A1 antibody to the tubulin inhibitor monomethylauristatin F (MMAF) via a maleimidocaproyl linker. A1mcMMAF exhibited potent in vivo antitumor activity in a variety of tumor models and induced long-term regressions for up to 100 days after the last dose. Strikingly, animals showed pathologic complete response in each model with doses as low as 3 mg antibody/kg dosed every 4 days. In a non-small cell lung cancer patient-derived xenograft model, in which 5T4 is preferentially expressed on the less differentiated tumor cells, A1mcMMAF treatment resulted in sustained tumor regressions and reduced TIC frequency. These results highlight the potential of ADCs that target the most aggressive cell populations within tumors, such as TICs. In exploratory safety studies, A1mcMMAF exhibited no overt toxicities when administered to cynomolgus monkeys at doses up to 10 mg antibody/kg/cycle × 2 and displayed a half-life of 5 days. The preclinical efficacy and safety data established a promising therapeutic index that supports clinical testing of A1mcMMAF.