Faculty Bibliography

The efficacy of prospective cancer treatments is routinely estimated by in vitro cell-line proliferation screens. However, it is unclear whether tumor aggressiveness and patient survival are influenced more by the proliferative or the migratory properties of cancer cells. To address this question, we experimentally measured proliferation and migration phenotypes across more than 40 breast cancer cell-lines. Based on the latter, we built and validated individual predictors of breast cancer proliferation and migration levels from the cells' transcriptomics. We then apply these predictors to estimate the proliferation and migration levels of more than 1000 TCGA breast cancer tumors. Reassuringly, both estimates increase with tumor's aggressiveness, as qualified by its stage, grade, and subtype. However, predicted tumor migration levels are significantly more strongly associated with patient survival than the proliferation levels. We confirmed these findings by conducting siRNA knock-down experiments on the highly migratory MDA-MB-231 cell lines and deriving gene knock-down based proliferation and migration signatures. We show that cytoskeletal drugs might be more beneficial in patients with high predicted migration levels. Taken together, these results testify to the importance of migration levels in determining patient survival.

Immunotherapy has shown modest activity in metastatic breast cancer (MBC). In this phase I dose escalation study, we assessed safety of tremelimumab, a humanized anti-CTLA4 monoclonal antibody, at starting dose 3 mg/kg, on the third day of palliative radiotherapy (2000cGy in 5 daily fractions) in patients with MBC. Primary objective was to determine the maximum tolerated dose (MTD) of tremelimumab combined with RT. Secondary objective was to assess response. Among 6 patients enrolled between July 2010 and October 2011, 5 had hormone receptor-positive MBC, 1 had triple negative MBC. Median age was 45 years. Common toxicities included lymphopenia (83%), fatigue (50%) and rash (33%). One dose-limiting toxicity occurred at 6 mg/kg, however the trial closed before MTD could be determined. One patient discontinued treatment due to a pathological fracture. Best response was stable disease (SD), 1 patient had SD for >6 months. Median follow up was 27.0 months. Median OS was 50.8 months, with 1 patient surviving >8 years. Peripheral blood mononuclear cell (PBMC) profiles showed increasing proliferating (Ki67+) Treg cells 1 week post treatment in 5 patients. Overall, tremelimumab at 3 mg/kg combined with RT appears to be a tolerable treatment strategy. Further studies are needed to optimize this combination approach.

Ovarian cancer (OC) is a heterogeneous disease usually diagnosed at a late stage. Experimental in vitro models that faithfully capture the hallmarks and tumor heterogeneity of OC are limited and hard to establish. We present a protocol that enables efficient derivation and long-term expansion of OC organoids. Utilizing this protocol, we have established 56 organoid lines from 32 patients, representing all main subtypes of OC. OC organoids recapitulate histological and genomic features of the pertinent lesion from which they were derived, illustrating intra- and interpatient heterogeneity, and can be genetically modified. We show that OC organoids can be used for drug-screening assays and capture different tumor subtype responses to the gold standard platinum-based chemotherapy, including acquisition of chemoresistance in recurrent disease. Finally, OC organoids can be xenografted, enabling in vivo drug-sensitivity assays. Taken together, this demonstrates their potential application for research and personalized medicine.

RASopathies are a family of related syndromes caused by mutations in regulators of the RAS/Extracellular Regulated Kinase 1/2 (ERK1/2) signaling cascade that often result in neurological deficits. RASopathy mutations in upstream regulatory components, such as NF1, PTPN11/SHP2, and RAS have been well-characterized, but mutation-specific differences in the pathogenesis of nervous system abnormalities remain poorly understood, especially those involving mutations downstream of RAS. Here, we assessed cellular and behavioral phenotypes in mice expressing a Raf1L613V gain-of-function mutation associated with the RASopathy, Noonan Syndrome. We report that Raf1L613V/wt mutants do not exhibit a significantly altered number of excitatory or inhibitory neurons in the cortex. However, we observed a significant increase in the number of specific glial subtypes in the forebrain. The density of GFAP+ astrocytes was significantly increased in the adult Raf1L613V/wt cortex and hippocampus relative to controls. OLIG2+ oligodendrocyte progenitor cells were also increased in number in mutant cortices, but we detected no significant change in myelination. Behavioral analyses revealed no significant changes in voluntary locomotor activity, anxiety-like behavior, or sociability. Surprisingly, Raf1L613V/wt mice mutants performed better than controls in select aspects of the water radial-arm maze, Morris water maze, and cued fear conditioning tasks. Overall, these data show that increased astrocyte and oligodendrocyte progenitor cell (OPC) density in the cortex coincides with enhanced cognition in Raf1L613V/wt mutants and further highlight the distinct effects of RASopathy mutations on nervous system development and function.

High-grade serous ovarian carcinoma (HGSC) is the most common and lethal subtype of gynecologic malignancy in women. The current standard of treatment combines cytoreductive surgery and chemotherapy. Despite the efficacy of initial treatment, most patients develop cancer recurrence, and 70% of patients die within 5 years of initial diagnosis. CA125 is the current FDA-approved biomarker used in the clinic to monitor response to treatment and recurrence, but its impact on patient survival is limited. New strategies for the discovery of HGSC biomarkers are urgently needed. Here, we describe a proteomics strategy to detect tumor-associated proteins in serum of HGSC patient-derived xenograft models. We demonstrate proof-of-concept applicability using two independent, longitudinal serum cohorts from HGSC patients.

Conditional knockout (KO) mouse models are invaluable for elucidating the physiological roles of platelets. The Pf4-Cre transgenic mouse is the current model of choice for generating megakaryocyte/platelet-specific KO mice. Platelets and leukocytes work closely together in a wide range of disease settings, yet the specific contribution of platelets to these processes remains unclear. This is partially due to the Pf4-Cre transgene being expressed in a variety of leukocyte populations. To overcome this issue, we developed a Gp1ba-Cre transgenic mouse strain in which Cre expression in driven by the endogenous Gp1ba locus. By crossing Gp1ba-Cre and Pf4-Cre mice to the mT/mG dual-fluorescence reporter mouse and performing a head-to-head comparison, we demonstrate more stringent megakaryocyte lineage-specific expression of the Gp1ba-Cre transgene. Broader tissue expression was observed with the Pf4-Cre transgene, leading to recombination in many hematopoietic lineages, including monocytes, macrophages, granulocytes, dendritic, B and T cells. Direct comparison of phenotypes of Csk, Shp1 or CD148 conditional KO mice generated using either the Gp1ba-Cre or Pf4-Cre strains revealed similar platelet phenotypes. However, additional inflammatory and immunological anomalies were observed in Pf4-Cre-generated KO mice due to non-specific deletion in other hematopoietic lineages. By excluding leukocyte contributions to phenotypes, the Gp1ba-Cre mouse will advance our understanding of the role of platelets in inflammation and other pathophysiological processes where platelet-leukocyte interactions are involved.

Hereditary leiomyomatosis and renal cell carcinoma (HLRCC) is an inherited cancer syndrome associated with a highly aggressive form of type 2 papillary renal cell carcinoma (PRCC). Germline inactivating alterations in fumarate hydratase (FH) cause HLRCC and result in elevated levels of reactive oxygen species (ROS). Recent work indicates that FH-/- PRCC cells have increased activation of ABL1, which promotes tumor growth, but how ABL1 is activated remains unclear. Given that oxidation can regulate protein-tyrosine phosphatase (PTP) catalytic activity, inactivation of an ABL-directed PTP by ROS might account for ABL1 activation in this malignancy. Our group previously developed "q-oxPTPome", a method that globally monitors the oxidation of classical PTPs. In this study, we present a refined q-oxPTPome, increasing its sensitivity by >10X. Applying q-oxPTPome to FH-deficient cell models showed that multiple PTPs were either highly oxidized (including PTPN12) or overexpressed. Highly oxidized PTP were those with relatively high sensitivity to exogenous H2O2. Most PTP oxidation in FH-deficient cells was reversible, although nearly 40% of PTPN13 was irreversibly oxidized to the sulfonic acid state. Using substrate-trapping mutants, we mapped PTPs to their putative substrates and found that only PTPN12 could target ABL1. Furthermore, knockdown experiments identified PTPN12 as the major ABL1 phosphatase, and overexpression of PTPN12 inhibited ABL1 phosphorylation and HLRCC cell growth. These results show that ROS-induced oxidation of PTPN12 accounts for ABL1 phosphorylation in HLRCC-associated PRCC, revealing a novel mechanism for inactivating a tumor suppressor gene product and establishing a direct link between pathological PTP oxidation and neoplastic disease.

Due to the involvement of SHP2 (SH2 domain-containing protein-tyrosine phosphatase) in human disease, including Noonan syndrome and cancer, several inhibitors targeting SHP2 have been developed. Here, we report that the commonly used SHP2 inhibitor NSC-87877 does not exhibit robust inhibitory effects on growth factor-dependent MAPK (mitogen-activated protein kinase) pathway activation and that the recently developed active site-targeting SHP2 inhibitors IIB-08, 11a-1, and GS-493 show off-target effects on ligand-evoked activation/trans-phosphorylation of the PDGFRβ (platelet-derived growth factor receptor β). GS-493 also inhibits purified human PDGFRβ and SRC in vitro, whereas PDGFRβ inhibition by IIB-08 and 11a-1 occurs only in the cellular context. Our results argue for extreme caution in inferring specific functions for SHP2 based on studies using these inhibitors.

Adaptive resistance to MEK inhibitors (MEK-Is) typically occurs via induction of genes for different receptor tyrosine kinases (RTKs) and/or their ligands, even in tumors of the same histotype, making combination strategies challenging. SHP2 (PTPN11) is required for RAS/ERK pathway activation by most RTKs, and might provide a common resistance node. We found that combining the SHP2 inhibitor SHP099 with a MEK-I inhibited the proliferation of multiple cancer cell lines in vitro. PTPN11 knockdown/MEK-I treatment had similar effects, while expressing SHP099 binding-defective PTPN11 mutants conferred resistance, demonstrating that SHP099 is on-target. SHP099/trametinib was highly efficacious in xenograft and/or genetically engineered models of KRAS-mutant pancreas, lung, and ovarian cancer and in wild type RAS-expressing triple negative breast cancer. SHP099 inhibited activation of KRAS mutants with residual GTPase activity, impeded SOS/RAS/MEK/ERK1/2 reactivation in response to MEK-Is and blocked ERK1/2-dependent transcriptional programs. We conclude that SHP099/MEK-I combinations could have therapeutic utility in multiple malignancies.

Vitamin C is an essential dietary requirement for humans. In addition to its known role as an antioxidant, vitamin C is a cofactor for Fe²⁺- and α-ketoglutarate-dependent dioxygenases (Fe²⁺/α-KGDDs) which comprise a large number of diverse enzymes, including collagen prolyl hydroxylases and epigenetic regulators of histone and DNA methylation. Vitamin C can modulate embryonic stem cell (ESC) function, enhance reprogramming of fibroblasts to induced pluripotent stem cells (iPSCs), and hinder the aberrant self-renewal of hematopoietic stem cells (HSCs) through its ability to enhance the activity of either Jumonji C (JmjC) domain-containing histone demethylases or ten-eleven translocation (TET) DNA hydroxylases. Given that epigenetic dysregulation is a known driver of malignancy, vitamin C may play a novel role as an epigenetic anticancer agent.