Irem Ozkan-Dagliyan’s research while affiliated with University of North Carolina at Chapel Hill and other places

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Publications (12)


Combination Therapies with CDK4/6 Inhibitors to Treat KRAS-mutant Pancreatic Cancer
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November 2022

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143 Reads

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74 Citations

Cancer Research

Craig M Goodwin

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Jennifer E Klomp

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Mutational loss of CDKN2A (encoding p16INK4A) tumor-suppressor function is a key genetic step that complements activation of KRAS in promoting the development and malignant growth of pancreatic ductal adenocarcinoma (PDAC). However, pharmacologic restoration of p16INK4A function with inhibitors of CDK4 and CDK6 (CDK4/6) has shown limited clinical efficacy in PDAC. Here, we found that concurrent treatment with both a CDK4/6 inhibitor (CDK4/6i) and an ERK–MAPK inhibitor (ERKi) synergistically suppresses the growth of PDAC cell lines and organoids by cooperatively blocking CDK4/6i-induced compensatory upregulation of ERK, PI3K, antiapoptotic signaling, and MYC expression. On the basis of these findings, a Phase I clinical trial was initiated to evaluate the ERKi ulixertinib in combination with the CDK4/6i palbociclib in patients with advanced PDAC (NCT03454035). As inhibition of other proteins might also counter CDK4/6i-mediated signaling changes to increase cellular CDK4/6i sensitivity, a CRISPR-Cas9 loss-of-function screen was conducted that revealed a spectrum of functionally diverse genes whose loss enhanced CDK4/6i growth inhibitory activity. These genes were enriched around diverse signaling nodes, including cell-cycle regulatory proteins centered on CDK2 activation, PI3K–AKT–mTOR signaling, SRC family kinases, HDAC proteins, autophagy-activating pathways, chromosome regulation and maintenance, and DNA damage and repair pathways. Novel therapeutic combinations were validated using siRNA and small-molecule inhibitor–based approaches. In addition, genes whose loss imparts a survival advantage were identified (e.g., RB1, PTEN, FBXW7), suggesting possible resistance mechanisms to CDK4/6 inhibition. In summary, this study has identified novel combinations with CDK4/6i that may have clinical benefit to patients with PDAC. Significance CRISPR-Cas9 screening and protein activity mapping reveal combinations that increase potency of CDK4/6 inhibitors and overcome drug-induced compensations in pancreatic cancer.


Targeting the ERK mitogen-activated protein kinase cascade for the treatment of KRAS-mutant pancreatic cancer

January 2022

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40 Reads

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13 Citations

Advances in Cancer Research

Mutational activation of the KRAS oncogene is found in ~ 95% of pancreatic ductal adenocarcinoma (PDAC), the major form of pancreatic cancer. With substantial experimental evidence that continued aberrant KRAS function is essential for the maintenance of PDAC tumorigenic growth, the National Cancer Institute has identified the development of effective anti-KRAS therapies as one of four major initiatives for pancreatic cancer research. The recent clinical success in the development of an anti-KRAS therapy targeting one specific KRAS mutant (G12C) supports the significant potential impact of anti-KRAS therapies. However, KRASG12C mutations comprise only 2% of KRAS mutations in PDAC. Thus, there remains a dire need for additional therapeutic approaches for targeting the majority of KRAS-mutant PDAC. Among the different directions currently being pursued for anti-KRAS drug development, one of the most promising involves inhibitors of the key KRAS effector pathway, the three-tiered RAF-MEK-ERK mitogen-activated protein kinase (MAPK) cascade. We address the promises and challenges of targeting ERK MAPK signaling as an anti-KRAS therapy for PDAC. In particular, we also summarize the key role of the MYC transcription factor and oncoprotein in supporting ERK-dependent growth of KRAS-mutant PDAC.


Figure 6. Concurrent Inhibition of Compensatory Signaling Enhances RAFi/ERKi Growth Inhibition (A) Pa02C and Pa14C cells were treated with RAFi (0.3 mM), ERKi (0.04 mM), FRAX597 (PAKi, 1 mM), or MK2206 (AKTi, 0.6 mM) alone or in combination (slash indicates concurrent inhibition; plus sign indicates sequential inhibition, inhibitor addition after 72 h). Remaining cells were stained with crystal violet after a total of 5 days. Data are the mean average of two independent experiments. Error bars are shown as ± SEM. (B) Pa02C and Pa14C cells were treated as in (A) for a total of 5 days. Cell lysates were immunoblotted to determine the levels of the indicated proteins. Data are representative of two independent experiments.
Low-Dose Vertical Inhibition of the RAF-MEK-ERK Cascade Causes Apoptotic Death of KRAS Mutant Cancers
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  • Full-text available

June 2020

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210 Reads

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79 Citations

Cell Reports

We address whether combinations with a pan-RAF inhibitor (RAFi) would be effective in KRAS mutant pancreatic ductal adenocarcinoma (PDAC). Chemical library and CRISPR genetic screens identify combinations causing apoptotic anti-tumor activity. The most potent combination, concurrent inhibition of RAF (RAFi) and ERK (ERKi), is highly synergistic at low doses in cell line, organoid, and rat models of PDAC, whereas each inhibitor alone is only cytostatic. Comprehensive mechanistic signaling studies using reverse phase protein array (RPPA) pathway mapping and RNA sequencing (RNA-seq) show that RAFi/ERKi induced insensitivity to loss of negative feedback and system failures including loss of ERK signaling, FOSL1, and MYC; shutdown of the MYC transcriptome; and induction of mesenchymal-to-epithelial transition. We conclude that low-dose vertical inhibition of the RAF-MEK-ERK cascade is an effective therapeutic strategy for KRAS mutant PDAC.

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Abstract B31: Combination inhibitor strategies targeting KRAS effector signaling in KRAS-mutant pancreatic cancer

May 2020

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16 Reads

Molecular Cancer Research

With KRAS mutations found in 95% of pancreatic ductal adenocarcinoma (PDAC), an effective anti-KRAS therapeutic strategy is anticipated to make a significant impact on the treatment of PDAC. Among the major directions currently being pursued, inhibitors of KRAS effector signaling are believed to be the most promising. However, as monotherapy, effector inhibitors have not demonstrated significant activity and concerns with normal tissue toxicity remain. Combination inhibitor strategies are considered the best approach to overcome these limitations. To identify effector inhibitor-based combinations, we applied a 525-oncology drug screen to identify combinations that enhanced the cytotoxic activity of inhibitors of the RAF-MEK-ERK and the PI3K-AKT-mTOR effector pathways. While many cytotoxic combinations were identified with RAF effector pathway inhibitors, few were identified with PI3K pathway inhibitors. The same classes of inhibitors were identified with RAF, MEK and ERK inhibitors. In addition to inhibitors of the PI3K-AKT-mTOR pathway, microtubule, HDAC and HSP90 inhibitors synergistically enhanced RAF and ERK inhibitor antitumor activity. Addressing a basis for synergy, we found that the different combinations showed enhanced loss of MYC protein, a key ERK substrate. In summary, our studies identify promising effector inhibitor-based combinations for PDAC treatment. Citation Format: Irem Ozkan-Dagliyan, Craig M. Goodwin, Kirsten L. Bryant, Samuel D. George, Kelly Lucas, Prson Gautam, Krister Wennerberg, Adrienne D. Cox, Channing J. Der. Combination inhibitor strategies targeting KRAS effector signaling in KRAS-mutant pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr B31.


Figure 4. KRAS Regulates MYC Protein Stability In Part Independently of FBXW7 and MYC Residues T58 and S62
Figure 5. Kinase Activation and MYC Protein Degradation Assays Identify a Role for MEK5-ERK5 Signaling in KRAS Regulation of MYC Protein Degradation (A) MIB/MS whole-kinome profiling upon transient siRNA KRAS knockdown in PDAC cells. Cell lysates were prepared from Panc 10.05 cells transfected with NS or KRAS 1
Figure 6. Identification of the MEK5-ERK5 Pathway as a Regulator of MYC Protein Stability in PDAC Cells
Figure 7. Concurrent ERK5 and ERK1/2 Inhibition Synergistically Destabilizes MYC, Reduces MYC S62 Phosphorylation and Inhibits PDAC Cell Growth (A) MIA PaCa-2 cells were transfected with NS or KRAS siRNA for 20 hr and treated with MG132 for an additional 6 hr. Cell lysates (Input) were used for immunoprecipitation (IP) of ERK5 and the amount of co-precipitated MYC was detected by immunoblotting (WB). (B) MIA PaCa-2 cells were treated with the ERK1/2i SCH772984 for the indicated times. Cell lysates were immunoblotted for total or phospho-specific antibodies (pEGFR, Y1068; pHER2 Y1248; pSRC, Y416).
KRAS Suppression-Induced Degradation of MYC Is Antagonized by a MEK5-ERK5 Compensatory Mechanism

November 2018

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335 Reads

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138 Citations

Cancer Cell

Our recent ERK1/2 inhibitor analyses in pancreatic ductal adenocarcinoma (PDAC) indicated ERK1/2-independent mechanisms maintaining MYC protein stability. To identify these mechanisms, we determined the signaling networks by which mutant KRAS regulates MYC. Acute KRAS suppression caused rapid proteasome-dependent loss of MYC protein, through both ERK1/2-dependent and -independent mechanisms. Surprisingly, MYC degradation was independent of PI3K-AKT-GSK3β signaling and the E3 ligase FBWX7. We then established and applied a high-throughput screen for MYC protein degradation and performed a kinome-wide proteomics screen. We identified an ERK1/2-inhibition-induced feedforward mechanism dependent on EGFR and SRC, leading to ERK5 activation and phosphorylation of MYC at S62, preventing degradation. Concurrent inhibition of ERK1/2 and ERK5 disrupted this mechanism, synergistically causing loss of MYC and suppressing PDAC growth.


The split energy and SPELL. a The target protein is computationally split, and the summed energy of the split parts (N and C lobes) are subtracted from the energy of the intact protein to calculate the split energy (SE). The split energy along with other filters, including solvent accessible area (SAA) and sequence conservation, were used to identify split sites. Prevention of spontaneous assembly is achieved using insertable FKBP (iFKBP), which destabilizes one of the lobes. Rapamycin or its photoactivatable analog produces both reassembly and correct folding of the destabilized lobe. b Proteins that have successful split sites between cores (labeled as “C”). Arrows show split sites described in the literature. Green arrows were described as successful, red as unsuccessful. Dashed boxes indicate known domains. c Proteins that have successful split sites at a secondary core. d A protein that has no clear separation between its cores
Lyn SPELL. a The structure of Lyn with split sites shown in red. b Based on the SPELL algorithm, we selected sites to test, including the promising residues 268 and 279, substantially higher in split energy than the cores at 1 and 2. 393 is a previously reported split site. c Phosphotyrosine blot of cell lysates with Lyn analogs split at N-lobe of the kinase domain. d Phosphotyrosine blot of cell lysates, including Lyn analogs split at C-lobe of the kinase domain. GFP is fused to the C terminus of Lyn to show the expression of full-length Lyn or C-lobe split protein
GDI SPELL. a The structure of RhoGDI bound to the GTPase Cdc42. b The SPELL algorithm indicated residue 66 as a split site. c The inhibitory activity of GDI SPELL (split at 66) was activated by rapamycin, whereas another design (split at 84) split at a small well did not display full activity with rapamycin. Error bars represent ± s.e.m. (n = 3) from three independent cell populations
Activation of Vav2 SPELL leads to cell protrusion. a Split energy profile of the DH domain of Vav2. Green arrow shows the least destabilized region and chosen loop for splitting. cons = sequence conservation, saa = surface exposure. b A structural model of Vav2 SPELL. iFKBP (light gray) was fused to the C terminus of the N-lobe of the DH domain (green), and FRB (dark gray) was fused to the N terminus of the C-lobe of the DH domain (blue) in the presence of rapamycin (purple). c A dual chain Rac1 FRET sensor was used to test Vav2 analogs. The FRET ratio (reflecting the activity of Rac1) with respect to the amount of mCherry-labeled Vav2 proteins: spVav-FKBP12-FRB = split protein generated using FKBP rather than iFKBP (mCherry-DHN-FKBP12 and FRB-DHC-PH-ZnF), Vav2 SPELL (mCherry-DHN-iFKBP and FRB- DHC-PH-ZnF), spVav-FRB = split protein made with no FKBP (mCherry-DHN and FRB- DHC-PH-ZnF), and Vav2 (mCherry-DH-PH-ZnF). d Vav2 SPELL activated with rapamycin or caged rapamycin, assayed as in c. e A HeLa cell expressing Vav2 SPELL, showing protrusion (green) and retraction (red) 19 min after rapamycin addition (upper left). f, g, h Morphology parameters (area, protrusive activity, and polarization index) of cells expressing Vav2 SPELL (green, mean ± s.e.m., n = 19 cells) vs. cells expressing only membrane marker (black, mean ± s.e.m., n = 36 cells). Rapamycin was added at 30 min (red line)
Computational design of chemogenetic and optogenetic split proteins

October 2018

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380 Reads

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84 Citations

Controlling protein activity with chemogenetics and optogenetics has proven to be powerful for testing hypotheses regarding protein function in rapid biological processes. Controlling proteins by splitting them and then rescuing their activity through inducible reassembly offers great potential to control diverse protein activities. Building split proteins has been difficult due to spontaneous assembly, difficulty in identifying appropriate split sites, and inefficient induction of effective reassembly. Here we present an automated approach to design effective split proteins regulated by a ligand or by light (SPELL). We develop a scoring function together with an engineered domain to enable reassembly of protein halves with high efficiency and with reduced spontaneous assembly. We demonstrate SPELL by applying it to proteins of various shapes and sizes in living cells. The SPELL server (spell.dokhlab.org) offers an automated prediction of split sites.




Evaluation of the selectivity and sensitivity of isoform-And mutation-specific RAS antibodies

September 2017

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85 Reads

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54 Citations

Science Signaling

There is intense interest in developing therapeutic strategies for RAS proteins, the most frequently mutated oncoprotein family in cancer. Development of effective anti-RAS therapies will be aided by the greater appreciation of RAS isoform–specific differences in signaling events that support neoplastic cell growth. However, critical issues that require resolution to facilitate the success of these efforts remain. In particular, the use of well-validated anti-RAS antibodies is essential for accurate interpretation of experimental data. We evaluated 22 commercially available anti-RAS antibodies with a set of distinct reagents and cell lines for their specificity and selectivity in recognizing the intended RAS isoforms and mutants. Reliability varied substantially. For example, we found that some pan- or isoform-selective anti-RAS antibodies did not adequately recognize their intended target or showed greater selectivity for another; some were valid for detecting G12D and G12V mutant RAS proteins in Western blotting, but none were valid for immunofluorescence or immunohistochemical analyses; and some antibodies recognized nonspecific bands in lysates from “Rasless” cells expressing the oncoprotein BRAFV600E. Using our validated antibodies, we identified RAS isoform–specific siRNAs and shRNAs. Our results may help to ensure the accurate interpretation of future RAS studies.


Some Novel Mannich Bases of 5-(3,4-Dichlorophenyl)-1,3,4-oxadiazole-2(3 H )-one and Their Anti-Inflammatory Activity: Novel Mannich Bases of 5-(3,4-Dichlorophenyl)-1,3,4-oxadiazole-2(3 H )-one

August 2017

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153 Reads

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21 Citations

Archiv der Pharmazie

Non-steroidal anti-inflammatory drugs (NSAIDs), which are widely used for the treatment of rheumatic arthritis, pain, and many different types of inflammatory disorders, cause serious gastrointestinal (GI) side effects. The free carboxylic acid group existing on their chemical structure is correlated with GI toxicity related with all routine NSAIDs. Replacing this functional group with the 1,3,4-oxadiazole bioisostere is a generally used strategy to obtain an anti-inflammatory agent devoid of GI side effects. In the present work, a novel group of 5-(3,4-dichlorophenyl)-1,3,4-oxadiazole-2(3H)-one Mannich bases were synthesized and characterized on the basis of IR, (1) H NMR, and elemental analysis results. The target compounds were first tested for cytotoxicity to determine a non-toxic concentration for anti-inflammatory screening. Anti-inflammatory effects of the compounds were evaluated by in vitro lipopolysaccharide (LPS)-induced NO production and in vivo carrageenan footpad edema with ulcerogenic profile. In LPS-induced RAW 264.7 macrophages, most of the compounds showed inhibitory activity on nitrite production while compounds 5a, 5h, and 5j exhibited the best profiles by suppressing the NO production. To evaluate the in vivo anti-inflammatory potency of the compounds, the inflammatory response was quantified by increment in paw size in the carrageenan footpad edema assay. The anti-inflammatory data scoring showed that compounds 5a-d, 5g, and 5j, at the dose of 100 mg/kg, exhibited anti-inflammatory activity, which for compound 5g was comparable to that of the reference drug indomethacin with 53.9% and 55.5% inhibition in 60 and 120 min, respectively.


Citations (9)


... inhibitors. CDK4/6 inhibitors synergized with the MAPK kinase (MEK) inhibitor in mutant RAS melanoma models 130 and with extracellular signal-regulated kinase (ERK) inhibitors in a mutant KRAS PDAC model 137 . In the same study, inhibition of CDK2 in addition to CDK4/6 resulted in increased apoptosis in pancreatic cancer cell lines compared to CDK4/6 inhibitor alone 137 . ...

Reference:

Resistance mechanisms and therapeutic strategies of CDK4 and CDK6 kinase targeting in cancer
Combination Therapies with CDK4/6 Inhibitors to Treat KRAS-mutant Pancreatic Cancer
  • Citing Article
  • November 2022

Cancer Research

... In PDAC the most common alterations are predominantly found in codon 12, specifically G12D, G12V, and G12R (13,14) and are often associated with poorer therapy response and overall survival (14). While G12D, G12V, and G12R mutations are the most frequent, G12R mutations have been linked to longer survival, particularly when not accompanied by PI3K pathway alterations (15). The development of small-molecule inhibitors targeting the KRAS pathway provides several treatment options for PDAC patients (16). ...

Targeting the ERK mitogen-activated protein kinase cascade for the treatment of KRAS-mutant pancreatic cancer
  • Citing Chapter
  • January 2022

Advances in Cancer Research

... Additionally, combination of IC 50 doses of mTORC1/2, PI3K and MEK inhibitors were also effective in preclinical models of ovarian clear cell carcinoma [305]. Also, in KRAS-mutant pancreatic cancer, concurrent inhibition of RAF and ERK was similarly highly synergistic in inducing apoptotic cell death at low doses, whereas each blocker alone was only cytostatic [306]. This low-dose multidrug approach to decrease driver network signaling has also been proven to be an effective strategy to suppress cancer metastasis [307]. ...

Low-Dose Vertical Inhibition of the RAF-MEK-ERK Cascade Causes Apoptotic Death of KRAS Mutant Cancers

Cell Reports

... A recent study published in Nature found that HCC patients with EGFR high were more likely to develop resistance to TKIs, particularly lenvatinib (12). Only in HCC patients with EGFR high , lenvatinib induces the feedback activation of EGFR and its downstream PAK2-ERK5 signaling pathway by inhibiting FGFR and downstream ERK1/2 (14), and simultaneously activates the downstream signaling pathway MEK1/2-ERK1/2, which is common with FGFR, resulting in strong proliferation ability of HCC cells while lenvatinib was administered. EGFR inhibitors effectively blocked feedback activation, and combined with Lenvatinib, produced synergistic antitumor effects, indicating that HCC patients with EGFR high could benefit from this combination (12,15). ...

KRAS Suppression-Induced Degradation of MYC Is Antagonized by a MEK5-ERK5 Compensatory Mechanism

Cancer Cell

... Potential sites of splitting can be computationally identified using the SPELL approach. 121 Single optogenetic or chemogenetic input signal Exploiting chemical (e.g., iFKBP-FRB-rapamycin) 120 or light (e.g., CRY2−CIB1) induced multimerization systems 103 ( Figure 3). ...

Computational design of chemogenetic and optogenetic split proteins

... As KRAS-mutant NSCLC tumors are extremely heterogenous, largely due to the diversity of their genetic alterations [9], comparing KRAS-mutant phenotypes using patient samples or NSCLC cell lines is challenging. Therefore, we began by using a panel of isogenic MEFs, initially engineered to become 'Ras-less' [30] and further genetically modified to express either KRAS WT , KRAS G12C or KRAS G12D [31]. Therefore, we could compare KRAS mutant isoforms directly without the confounding effects of co-mutations seen in lung tumors. ...

Evaluation of the selectivity and sensitivity of isoform-And mutation-specific RAS antibodies
  • Citing Article
  • September 2017

Science Signaling

... Inflammation is a defensive immune reaction that develops in our body against mechanical injury, pathogens or irritants. The healing process occurring in the body is initiated by inflammation after subjecting it to various physiological adaptations to minimize the tissue damage [17][18][19]. On the other hand, long-term inflammatory conditions are not very useful for the body and can lead to various disorders, like multiple sclerosis, atherosclerosis, retinitis, psoriasis, inflammatory bowel diseases, osteoarthritis and rheumatoid arthritis. ...

Some Novel Mannich Bases of 5-(3,4-Dichlorophenyl)-1,3,4-oxadiazole-2(3 H )-one and Their Anti-Inflammatory Activity: Novel Mannich Bases of 5-(3,4-Dichlorophenyl)-1,3,4-oxadiazole-2(3 H )-one

Archiv der Pharmazie

... KS15 has also been reported to disrupt the circadian system in mouse cells (via disrupted binding to BMAL1 [37,41,42], which is essential for circadian rhythmic transcriptional repression [9,10,16]). We therefore next extended our circadian reporter approach to mouse fibroblasts expressing the PER2::LUC translational reporter [54]. ...

Dual modes of CLOCK:BMAL1 inhibition mediated by Cryptochrome and Period proteins in the mammalian circadian clock

Genes & Development

... In darkness-grown gemmalings, the RFP-MpCRY fusion protein was located mainly in the cytosol and plasma membrane (Figs 4a, S5). After blue light exposure, RFP-MpCRY accumulation occurred almost entirely in a single large cell organellethe nucleusand typical nuclear bodies were formed (Figs 4a, S5), similar to what occurs with the nuclear bodies or photobodies that are the activated state of Arabidopsis CRY2 (Yu et al., 2009;Ozkan-Dagliyan et al., 2013;Wang et al., 2016), although levels of cytosol-localized RFP-MpCRY were low (Figs 4a, S5). However, red and far-red light did not affect the localization of RFP-MpCRY (Fig. S5). ...

Formation of Arabidopsis Cryptochrome 2 Photobodies in Mammalian Nuclei: Application as an Optogenetic DNA Damage Checkpoint Switch.
  • Citing Article
  • July 2013

Journal of Biological Chemistry