Richard Z. Liu

Mass Spectrometry Mapping of Epidermal Growth Factor Receptor Phosphorylation Related to Oncogenic Mutations and Tyrosine Kinase Inhibitor Sensitivity

The epidermal growth factor receptor (EGFR) plays an important role in cancer by activating downstream signals important in growth and survival. Inhibitors of EGFR are frequently selected as treatment for cancer including lung cancer. We performed an unbiased and comprehensive search for EGFR phosphorylation events related to somatic activating mutations and EGFR inhibitor (erlotinib) sensitivity. EGFR immunoprecipitation combined with high resolution liquid chromatography-mass spectrometry and label free quantitation characterized EGFR phosphorylation. Thirty (30) phosphorylation sites were identified including 12 tyrosine (pY), 12 serine (pS), and 6 threonine (pT). Site-specific phosphorylation was monitored by comparing ion signals from the corresponding unmodified peptide. Phosphorylation sites related to activating mutations in EGFR as well as sensitivity to erlotinib were identified using 31 lung cancer cell lines. We identified three sites (pY1092, pY1110, pY1172) correlated with activating mutations and three sites (pY1110, pY1172, pY1197) correlated with erlotinib sensitivity. Five sites (pT693, pY1092, pY1110, pY1172, and pY1197) were inhibited by erlotinib in concentration-dependent manner. Erlotinib sensitivity was confirmed using liquid chromatography coupled to multiple reaction monitoring (LC-MRM) and quantitative Western blotting. This LC-MS/MS strategy can quantitatively assess site-specific EGFR phosphorylation and can identify relationships between somatic mutations or drug sensitivity and protein phosphorylation.

A database of reaction monitoring mass spectrometry assays for elucidating therapeutic response in cancer

Purpose: The Quantitative Assay Database (QuAD), http://proteome.moffitt.org/QUAD/, facilitates widespread implementation of quantitative mass spectrometry in cancer biology and clinical research through sharing of methods and reagents for monitoring protein expression and modification.

Quantification of β-catenin signaling components in colon cancer cell lines, tissue sections, and microdissected tumor cells using reaction monitoring mass spectrometry

Reaction monitoring mass spectrometry has emerged as a powerful tool for targeted detection and quantification of proteins in clinical samples. Here, we report the use of gel electrophoresis for protein fractionation and liquid chromatography coupled to multiple reaction monitoring mass spectrometry (LC-MRM) screening for quantitative analysis of components from the Wnt/beta-catenin signaling pathway, which contributes to colon tumor formation and progression. In silico tools are used to design LC-MRM screens for each target protein. Following successful peptide detection, stable isotope labeled peptides are synthesized and developed as internal standards. Then, the assays are implemented in colon cancer cell lines to achieve detection in minimal amounts of cells, compatible with direct translation to clinical specimens. Selected assays are compared with qualitative results from immunoblotting (Westerns) and translated to individual frozen colon tissue sections and laser capture microdissected tumor cells. This LC-MRM platform has been translated from in vitro models to clinical specimens, forming the basis for future experiments in patient assessment.

JAK1 truncating mutations in gynecologic cancer define new role of cancer-associated protein tyrosine kinase aberrations

Cancer-associated protein tyrosine kinase (PTK) mutations usually are gain-of-function (GOF) mutations that drive tumor growth and metastasis. We have found 50 JAK1 truncating mutations in 36 of 635 gynecologic tumors in the Total Cancer Care® (TCC®) tumor bank. Among cancer cell lines containing JAK1 truncating mutations in the Cancer Cell Line Encyclopedia databank, 68% are gynecologic cancer cells. Within JAK1 the K142, P430, and K860 frame-shift mutations were identified as hot spot mutation sites. Sanger sequencing of cancer cell lines, primary tumors, and matched normal tissues confirmed the JAK1 mutations and showed that these mutations are somatic. JAK1 mediates interferon (IFN)-γ-regulated tumor immune surveillance. Functional assays show that JAK1 deficient cancer cells are defective in IFN-γ-induced LMP2 and TAP1 expression, loss of which inhibits presentation of tumor antigens. These findings identify recurrent JAK1 truncating mutations that could contribute to tumor immune evasion in gynecologic cancers, especially in endometrial cancer.

Quantification of peptides from immunoglobulin constant and variable regions by LC‐MRM MS for assessment of multiple myeloma patients

Quantitative MS assays for Igs are compared with existing clinical methods in samples from patients with plasma cell dyscrasias, for example, multiple myeloma (MM).

Coupling an EML4-ALK–centric interactome with RNA interference identifies sensitizers to ALK inhibitors

A network built around the kinase ALK reveals potential targets to enhance ALK inhibitor efficacy in some lung cancer patients. Networking ALK for combination therapies Some lung cancers have high activity of the kinase ALK as the result of rearrangements between the genes EML4 and ALK. ALK inhibitors are effective in some patients, but resistance to single-agent therapy is common. Using phosphoproteomics and an RNA interference screen, Zhang et al. derived a signaling network mediated by ALK in EML4-ALK–rearranged lung cancer cell lines. From this network, they identified many candidates that could sensitize cells to ALK inhibition. Indeed, knocking down either of two of these two proteins, the scaffolding proteins FRS2 and CC2D1A, sensitized cell lines to the ALK inhibitors crizotinib and alectinib. Thus, a clinical strategy that inhibits FRS2 or CC2D1A might enhance the efficacy of ALK inhibitors in some patients. Patients with lung cancers harboring anaplastic lymphoma kinase (ALK) gene fusions benefit from treatment with ALK inhibitors, but acquired resistance inevitably arises. A better understanding of proximal ALK signaling mechanisms may identify sensitizers to ALK inhibitors that disrupt the balance between prosurvival and proapoptotic effector signals. Using affinity purification coupled with mass spectrometry in an ALK fusion lung cancer cell line (H3122), we generated an ALK signaling network and investigated signaling activity using tyrosine phosphoproteomics. We identified a network of 464 proteins composed of subnetworks with differential response to ALK inhibitors. A small hairpin RNA screen targeting 407 proteins in this network revealed 64 and 9 proteins that when knocked down sensitized cells to crizotinib and alectinib, respectively. Among these, knocking down fibroblast growth factor receptor substrate 2 (FRS2) or coiled-coil and C2 domain–containing protein 1A (CC2D1A), both scaffolding proteins, sensitized multiple ALK fusion cell lines to the ALK inhibitors crizotinib and alectinib. Collectively, our data set provides a resource that enhances our understanding of signaling and drug resistance networks consequent to ALK fusions and identifies potential targets to improve the efficacy of ALK inhibitors in patients.

Abstract LB-140: Scaling discovery proteomics to large lung cancer cohorts using data independent acquisition

The success of label free protein quantification relies on larger cohort sizes to accurately profile protein expression patterns enabling specific targets to be translated to confirmatory studies. Thus, proteome profiling experiments must satisfy two primary requirements: maximizing breadth and depth of protein sampling as well as facilitating automated data processing. The approach presented here integrates discovery experiments used to create reference databases and rapid data independent acquisition (DIA) methods to examine large clinical cohorts using Samples used to evaluate the robustness of the study were collected from lung squamous cell carcinoma (LUSC) and lung adenocarcinoma (LUAD). A sample total of 159 (50 LUSC, 46 LUAD, and 63 adjacent control lung tissues) were analyzed using UHPLC separations coupled to a hybrid quadrupole-orbital ion trap mass spectrometer (QExactive Plus) for DIA. Automated data processing was performed by matching peptides in spectral libraries developed from expression proteomics experiments using pooled tissue proteomes. Data were evaluated to determine differences between LUSC, LUAD, and adjacent control tissue, which were then compared to previous literature to prioritize candidate biomarkers and evaluate the performance of DIA LC-MS/MS for tumor proteome profiling. Hierarchical clustering visualized potential tumor classification schemes based on proteomic phenotypes. In a further study, analysis of cores from a tissue microarray produced quantitative data for > 3,000 proteins per core, indicating that the technology can be applied to minimal amounts of formalin fixed paraffin embedded tumor tissue. Because TMAs are assembled to address specific clinical questions, further analysis of these cohorts is an extremely valuable use for DIA; tumor proteome profiles can be rapidly accumulated and mapped to the clinical variables used to select samples for the TMA. Data independent acquisition provides a method for discovery proteomics that balances a sufficient depth of coverage with the ability to analyze large cohorts of patients (n = 100-400) within 1-2 months on a single instrument. Initial data have been produced for lung squamous cell carcinoma and lung adenocarcinoma, which indicate its utility for assessment of patient groups. Citation Format: Scott Peterman, Bin Fang, Melissa Hoffmann, Amol Prakash, Paul A. Stewart, Richard Liu, Matthew Smith, Joseph Johnson, Steven Eschrich, Guolin Zhang, Eric Haura, John Koomen. Scaling discovery proteomics to large lung cancer cohorts using data independent acquisition. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-140.

Abstract A58: JAK1 deficiency as a novel mechanism of tumor immune escape in uterine cancer

Recognition of MHC class I antigens on the surface of tumor cells is an essential process in the effector phase of CD8+ cytotoxic T lymphocyte (CTL)-mediated anti-tumor response. Presentation of MHC class I antigens on tumor cells is regulated by the JAK1/JAK2-mediated interferon-γ signaling pathway. We analyzed the targeted exome sequencing data of 3,274 human tumors from 48 different tissues in the Total Cancer Care (TCC) tumor bank and found JAK1 truncating mutations in 36 of 635 gynecologic tumors. The highest JAK1 truncating mutation rate (9.5%) was found in endometrial cancer. These truncating mutations result in the loss of the JAK1 protein tyrosine kinase (PTK) domain that is located in the C-terminal region. JAK1 truncating mutations in cancer cell lines of the Cancer Cell Line Encyclopedia (CCLE) databank also occur most often in endometrial cancer cells. Analysis of mutation sites identified three mutation hot spots. Re-sequencing of cancer cell lines, primary tumors, and matched normal tissues confirmed JAK1 mutations and revealed that these JAK1 mutations are somatic. Besides JAK1, recurrent JAK2 loss-of-function (LOF) mutations were also found in TCC tumors but at lower rates. Similar to JAK1, the highest rate of LOF JAK2 mutations was found in endometrial/uterine cancer. No JAK1 or JAK2 truncating mutation was identified in 130 samples from hematological malignancies. Functional assays showed that JAK1 deficient cancer cells were defective in interferon-γ-induced expression of tumor antigen processing machinery proteins LMP2 and TAP1 and cell surface expression of HLA molecules. These data identify recurrent JAK1 deficiency in endometrial cancer that impairs tumor antigen presentation by the MHC class I complex to CTLs. While PTKs have been perceived mostly as oncogenes and PTK inhibitors, including JAK inhibitors, have been developed for cancer therapy, our findings suggest that JAK1 is a novel tumor suppressor and that LOF JAK1 mutations allow tumor immune escape. Our findings also raise caution about the use of JAK inhibitors as therapeutic agents in solid tumors. Citation Format: Yuan Ren, Yonghong Zhang, Richard Z. Liu, David A. Fenstermacher, Kenneth L. Wright, Jamie K. Teer, Jie Wu. JAK1 deficiency as a novel mechanism of tumor immune escape in uterine cancer. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr A58.

Abstract 1560: Loss-of-function JAK1 mutations reveal a new role of protein tyrosine kinase mutations in human cancer

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Protein tyrosine kinases (PTKs) have been perceived mostly as proto-oncogenes. PTK mutations that have been studied in human cancer usually have increased kinase activities to drive malignant phenotypes. Thus, these PTK mutations are gain-of-function (GOF) mutations. By analyzing the targeted exome sequencing data of 3,274 human tumors from 48 different tissues in the Total Cancer Care (TCC) tumor bank, we have found JAK1 truncating mutations in 36 of 635 gynecologic tumors. The highest JAK1 truncating mutation rate (9.5%) was found in endometrial cancer. These truncating mutations result in the loss of the JAK1 PTK domain. Thus, they are loss-of-function (LOF) mutations. JAK1 truncating mutations in the Cancer Cell Line Encyclopedia (CCLE) databank also occur most often in endometrial cancer cell lines. Analysis of mutation sites identified three mutation hot spots. Re-sequencing of cancer cell lines, primary tumors, and matched normal tissues confirmed JAK1 mutations and revealed that these JAK1 mutations are somatic. JAK1 and JAK2 mediate interferon (IFN)-γ-regulated tumor immune surveillance. Recurrent JAK2 LOF mutations were also found in TCC tumors but at lower rates. Similar to JAK1, the highest rate of LOF JAK2 mutations was found in endometrial/uterine cancer. No JAK1 or JAK2 truncating mutation was identified in 130 samples from hematological malignancies. Functional assays show that JAK1-deficient cancer cells are defective in IFN-γ-induced expression of tumor antigen processing machinery proteins LMP2 and TAP1 and cell surface expression of HLA molecules. These data identify recurrent JAK1 deficiency in endometrial cancer that could impair tumor antigen presentation by the MHC class I complex to cytotoxic T lymphocytes. Our findings suggest that JAK1 is a PTK tumor suppressor and reveal a novel mechanism of tumor immune evasion via LOF mutations in a PTK. Citation Format: Yuan Ren, Yonghong Zhang, Richard Z. Liu, David A. Fenstermacher, Kenneth L. Wright, Jamie K. Teer, Jie Wu. Loss-of-function JAK1 mutations reveal a new role of protein tyrosine kinase mutations in human cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1560. doi:10.1158/1538-7445.AM2014-1560

Abstract LB-234: Suppression of Bruton tyrosine kinase with X-022, a highly selective next-generation irreversible inhibitor

BTK is a member of the TEC family of kinases and is a critical component of B cell receptor (BCR) signaling, promoting B cell maturation, survival and proliferation. BCR signaling is requisite in all B cell-mediated malignancies where clinical trials with BTK inhibitors have proven efficacious. X-Rx utilized its drug discovery platform and knowhow to advance multiple novel series of BTK inhibitors. X-022 was then identified as a next generation, oral small molecule BTK inhibitor. X-022 irreversibly inhibits BTK with an IC50 of 2.3 nM. In primary human B cell and HWB assays, X-022 inhibited B cell activity with IC50 values of 49 and 93 nM, respectively. X-022 showed superior selectivity against a panel of 456 kinases, only inhibiting at >90% BTK at a concentration of 1000 nM. Oral bioavailability of >20% is observed in mice, rats and dogs. In a mouse PK/PD model, X-022 showed a dose dependent decrease in B cell activation measured over an 8 hour time interval with >98% inhibition observed at 10 mg/kg po. In an established model of collagen induced arthritis in mice, X-022 caused a dose dependent decrease in disease severity consistent with mechanism of action. No safety issues were identified in a pilot study in rodents at doses up to 300 mg/kg po. X-022 is a promising next generation irreversible oral BTK inhibitor for the treatment of B cell malignancies and chronic autoimmune diseases. In addition, the high degree of selectivity combined with favorable molecular properties make X-022 an excellent candidate for co-formulation with other targeted therapeutics. Citation Format: Mark J. Mulvihill, Xiangyang Chen, JP Shaw, Richard Liu, Haihong Ni, Lee Babiss, Louis Renzetti. Suppression of Bruton tyrosine kinase with X-022, a highly selective next-generation irreversible inhibitor. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-234. doi:10.1158/1538-7445.AM2014-LB-234