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The transforming growth factor β (TGFβ) pathway orchestrates an extensive transcriptional program that is important for many processes in the cell. For example, TGFβ regulates cell cycle, migration, and epithelial-to-mesenchymal transition. The TGFβ pathway has a dual role in cancer: it is involved in early-stage tumor suppression but also contributes to tumor progression by promoting invasion. To identify the novel genes involved in TGFβ pathway signaling, we have performed a functional genetic loss-of-function screen. We screened a small interfering RNA library targeting 700 kinases and kinase-related genes in a TGFβ-responsive reporter assay. Several genes were identified that upon knockdown could repress the reporter signal; among these are the two cellular receptors for TGFβ. In addition to these two known components of the TGFβ pathway, several genes were identified that were previously not linked to the TGFβ signaling. Knockdown of one of these genes, the IRAK2 kinase, resulted not only in an impaired TGFβ target gene response but also in a reduction of the nuclear accumulation and phosphorylation of SMAD2. In addition, suppression of interleukin-1R–associated kinase 2 expression led to a partial override of a TGFβ-induced cell cycle arrest. Our data show that interleukin-1R–associated kinase 2 is a novel and critical component of TGFβ signaling. Mol Cancer Res; 8(4); 592–603. ©2010 AACR.

Supplementary Data from Interleukin-1R–Associated Kinase 2 Is a Novel Modulator of the Transforming Growth Factor β Signaling Cascade

Cancer homeostasis depends on a balance between activated oncogenic pathways driving tumorigenesis and engagement of stress-response programs that counteract the inherent toxicity of such aberrant signaling. While inhibition of oncogenic signaling pathways has been explored extensively, there is increasing evidence that overactivation of the same pathways can also disrupt cancer homeostasis and cause lethality. We show here that inhibition of Protein Phosphatase 2A (PP2A) hyperactivates multiple oncogenic pathways and engages stress responses in colon cancer cells. Genetic and compound screens identify combined inhibition of PP2A and WEE1 as synergistic in multiple cancer models by collapsing DNA replication and triggering premature mitosis followed by cell death. This combination also suppressed the growth of patient-derived tumorsin vivo. Remarkably, acquired resistance to this drug combination suppressed the ability of colon cancer cells to form tumorsin vivo. Our data suggest that paradoxical activation of oncogenic signaling can result in tumor suppressive resistance.

Intratumoral heterogeneity has been described for various tumor types and models of human cancer, and can have profound effects on tumor progression and drug resistance. This study describes an in-depth analysis of molecular and functional heterogeneity among subclonal populations (SCPs) derived from a single triple-negative breast cancer cell line, including copy number analysis, whole-exome and RNA sequencing, proteome analysis, and barcode analysis of clonal dynamics, as well as functional assays. The SCPs were found to have multiple unique genetic alterations and displayed significant variation in anchorage independent growth and tumor forming ability. Analyses of clonal dynamics in SCP mixtures using DNA barcode technology revealed selection for distinct clonal populations in different in vitro and in vivo environmental contexts, demonstrating that in vitro propagation of cancer cell lines using different culture conditions can contribute to the establishment of unique strains. These analyses also revealed strong enrichment of a single SCP during the development of xenograft tumors in immune-compromised mice. This SCP displayed attenuated interferon signaling in vivo and reduced sensitivity to the antiproliferative effects of type I interferons. Reduction in interferon signaling was found to provide a selective advantage within the xenograft microenvironment specifically. In concordance with the previously described role of interferon signaling as tumor suppressor, these findings suggest that similar selective pressures may be operative in human cancer and patient-derived xenograft models.

Recently, organoid technology has been used to generate a large repository of breast cancer organoids. Here we present an extensive evaluation of the ability of organoid culture technology to preserve complex stem/progenitor and differentiated cell types via long-term propagation of normal human mammary tissues. Basal/stem and luminal progenitor cells can differentiate in culture to generate mature basal and luminal cell types, including ER+ cells that have been challenging to maintain in culture. Cells associated with increased cancer risk can also be propagated. Single-cell analyses of matched organoid cultures and native tissues by mass cytometry for 38 markers provide a higher resolution representation of the multiple mammary epithelial cell types in the organoids, and demonstrate that protein expression patterns of the tissue of origin can be preserved in culture. These studies indicate that organoid cultures provide a valuable platform for studies of mammary differentiation, transformation, and breast cancer risk., Organoid technology has enabled the generation of several breast cancer organoids. Here, the authors combine propagation of normal human mammary tissues with mass cytometry to evaluate the ability of organoid culture technologies to preserve stem cells and differentiated cell types.

The extent and importance of functional heterogeneity and crosstalk between tumor cells is poorly understood. Here, we describe the generation of clonal populations from a patient-derived ovarian clear cell carcinoma model which forms malignant ascites and solid peritoneal tumors upon intraperitoneal transplantation in mice. The clonal populations are engineered with secreted Gaussia luciferase to monitor tumor growth dynamics and tagged with a unique DNA barcode to track their fate in multiclonal mixtures during tumor progression. Only one clone, CL31, grows robustly, generating exclusively malignant ascites. However, multiclonal mixtures form large solid peritoneal metastases, populated almost entirely by CL31, suggesting that transient cooperative interclonal interactions are sufficient to promote metastasis of CL31. CL31 uniquely harbors ERBB2 amplification, and its acquired metastatic activity in clonal mixtures is dependent on transient exposure to amphiregulin, which is exclusively secreted by non-tumorigenic clones. Amphiregulin enhances CL31 mesothelial clearance, a prerequisite for metastasis. These findings demonstrate that transient, ostensibly innocuous tumor subpopulations can promote metastases via “hit-and-run” commensal interactions., Cooperative interactions among tumor cells may have important implications for metastasis. Here, the authors examined the spatio-temporal nature of interactions among clonal populations of ovarian carcinoma cells and found that transient interactions cells can promote metastases via commensal interactions.

To interrogate functional heterogeneity and crosstalk between tumor cells, we generated clonal populations from a patient-derived ovarian clear cell carcinoma model which forms malignant ascites and solid peritoneal tumors upon intraperitoneal transplantation in mice. The clonal populations were engineered with secreted Gaussia luciferase to monitor tumor growth dynamics and tagged with a unique DNA barcode to track their fate in multiclonal mixtures during tumor progression. Only one clone, CL31, grew robustly, generating exclusively malignant ascites. However, multiclonal mixtures formed large solid peritoneal metastases, populated almost entirely by CL31, suggesting that transient cooperative interclonal interactions were sufficient to promote metastasis of CL31. CL31 uniquely harbored ERBB2 amplification, and its acquired metastatic trait was dependent on transient exposure to amphiregulin, which was exclusively secreted by non-tumorigenic clones. Amphiregulin enhanced CL31 mesothelial clearance, a prerequisite for metastasis. These findings demonstrate that transient, ostensibly innocuous tumor subpopulations can promote metastases via “hit- and-run” commensal interactions.

Cancer-associated mutations that stabilize NRF2, an oxidant defense transcription factor, are predicted to promote tumor development. Here, utilizing 3D cancer spheroid models coupled with CRISPR-Cas9 screens, we investigate the molecular pathogenesis mediated by NRF2 hyperactivation. NRF2 hyperactivation was necessary for proliferation and survival in lung tumor spheroids. Antioxidant treatment rescued survival but not proliferation, suggesting the presence of distinct mechanisms. CRISPR screens revealed that spheroids are differentially dependent on the mammalian target of rapamycin (mTOR) for proliferation and the lipid peroxidase GPX4 for protection from ferroptosis of inner, matrix-deprived cells. Ferroptosis inhibitors blocked death from NRF2 downregulation, demonstrating a critical role of NRF2 in protecting matrix-deprived cells from ferroptosis. Interestingly, proteomics analyses show global enrichment of selenoproteins, including GPX4, by NRF2 downregulation, and targeting NRF2 and GPX4 killed spheroids overall. These results illustrate the value of spheroid culture in revealing environmental or spatial differential dependencies on NRF2 and reveal exploitable vulnerabilities of NRF2-hyperactivated tumors.

Intratumoral heterogeneity impacts both tumor progression and drug sensitivity. While intratumoral genetic heterogeneity has been extensively described, there is still little understanding of the extent of heterogeneity in drug sensitivity within a single tumor, the intra tumor dynamics of different cell populations during anti-cancer compound treatment, and the role of non-cell autonomous mechanism in drug resistance. The objective of this study is to characterize heterogeneity in drug sensitivity within a model for human breast cancer, identify interactions between clonal population influencing drug sensitivity, and use this to model the dynamics of sub populations in a tumor during anti-cancer compound treatment. We have characterized 31 subclonal cell populations (SCPs) that were generated by single cell cloning of the triple-negative breast cancer cell line MDA-MB-468. These SCPs display considerable phenotypic and molecular heterogeneity with respect to morphology, proliferation rate, RNA expression and tumorigenicity in mice. To examine the extent of heterogeneity in drug sensitivity, we designed and screened a 200-small molecule compound library in 24 SCPs and the parental cell line. We observed considerable heterogeneity in drug sensitivity and selected 36 compounds for validation in the SCPs that expressed the greatest range of sensitivity (determined by IC50). To investigate whether interactions between clonal population can influence drug sensitivity heterogeneously responsive subclonal cell lines were transduced with lentiviruses encoding for the H2B-GFP or H2B-RFP fusion protein and then cocultured in the absence or presence of different compounds. We observed shifts in drug sensitivity for SCPs that were cocultured with differentially responsive clones. Examples include SCPs 19 and 27 two highly sensitive populations that were protected by the presence of less sensitive SCPs when under gemcitabine treatment, resulting in IC50-values that were 20-fold higher then when cultured alone. RNA-sequencing and RPPA analysis will be used to characterize this non-cell autonomous mechanism of resistance and the relationship with identified transcriptional programs or genes which expression correlates with drug sensitivity. We are also generating and will evaluate predictive models for drug combination using the SCP drug sensitivity profiles. The results of this study show that there is considerable heterogeneity in drug sensitivity among clonal subpopulations derived from a single cancer cell line. Ongoing experiments will provide insight into the effect of drug combinations and unique treatment schedules on distinct tumor cell subpopulations and drug sensitivity within heterogenous mixtures. Moreover, we will be able to test for and investigate non-cell autonomous modes of drug resistance, which may guide the development of new drug combination treatments or schedules. Citation Format: Chandler M Friend, Joan S Brugge, Hendrik J Kuiken, Steven M Corsello, Christopher C Mader. Characterization of intratumoral heterogeneity in drug sensitivity and non-cell autonomous mechanisms of drug resistance using subclonal cell populations derived from a single breast cancer cell line [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A132. doi:10.1158/1535-7163.TARG-19-A132

Antioxidants have historically been thought of as cancer preventative agents; however, increasing evidence in recent years indicate that antioxidants promote cancer cell survival. Using 3D spheroid models, our lab previously demonstrated that normal cells die in the inner layers of spheroids, whereas cancer cells survive. Multiple lines of evidence indicate that survival of inner cells within tumor spheroids is dependent on escape from apoptosis and reactive oxygen species (ROS)-induced cell death through upregulation of anti-apoptotic and antioxidant programs. While the mechanisms associated with apoptosis are well-established, there is relatively little understood about the cellular processes involved in ROS-induced cell death (origins of ROS, types of oxidative stress experienced, mechanisms of cell death, antioxidants involved etc.). Tumor cells with mutations in the nuclear factor erythroid 2-related factor 2 (NRF2) pathway that lead to hyperactivation of this transcription factor serve as an excellent model to study the role of antioxidant programs in the regulation of survival processes. NRF2 is the master regulator of the antioxidant response directly regulating expression of antioxidant enzymes as well as proteins required for the production of non-enzymatic antioxidants. Furthermore, mutations in the NRF2 pathway are frequent in tumor types that are associated with oxidative stress such as lung cancer. We found that knock-down of NRF2 affected cell survival and growth in 3D by regulating (i) cell proliferation and (ii) inner cell survival. To better understand the specific processes responsible for ROS-induced cell death and the antioxidant program(s) utilized by cancer cells to bypass these death programs, we performed CRISPR screens in 3D using lung cancer cell lines with NRF2 hyperactivation to identify downstream targets of NRF2 and/or antioxidants that collaborate with NRF2 to regulate cell survival and growth in 3D. As predicted, we observed drop-out of sgRNAs targeting NRF2. Notably, a top drop-out hit was glutathione peroxidase 4 (GPX4), an essential antioxidant enzyme that reduces membrane phospholipid hydroperoxides to prevent a form of non-apoptotic cell death known as ferroptosis. We found that inhibition of GPX4 resulted in elevated levels of lipid peroxidation and death in the inner cells of 3D spheroids which were rescuable by the ferroptosis inhibitor Ferrostatin-1, implicating ferroptotic cell death in 3D biology. Surprisingly, knock-down of NRF2 resulted in stabilization of GPX4 at the post-transcriptional level. Finally, knock-down of NRF2 combined with GPX4 inhibition induced cell death in both inner and outer cells of 3D spheroids. These data suggest that cancer cells exploit the functional interplay between NRF2 and GPX4 to survive in high oxidative stress environments. This work was supported by funding from the ROADS Program funded by F. Hoffmann-La Roche Ltd.. Note: This abstract was not presented at the meeting. Citation Format: Patricia Cho, Nobuaki Takahashi, Laura M. Selfors, Hendrik J. Kuiken, Joan S. Brugge. Interplay between NRF2 and GPX4 is critical for cancer cell survival in 3D spheroids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 884.

Intratumoral heterogeneity can have profound effects on tumor progression and drug resistance. While tumor cell heterogeneity has been described at many levels, there is a poor understanding of the extent of heterogeneity in drug sensitivity within a single tumor, the dynamics of tumor cell subpopulations during treatment with anti-cancer agents and non-cell autonomous mechanisms of drug resistance. The overall objective of this study is to characterize heterogeneity in drug sensitivity within a model for human breast cancer and to model and evaluate the effect of drug combinations on the dynamics of subpopulations within heterogeneous mixtures. We have characterized 31 subclonal cell populations (SCPs) that were generated through single cell cloning of the triple-negative breast cancer cell line MDA-MB-468. These SCPs display considerable heterogeneity with respect to morphology, proliferation rate, colony formation in soft agar and tumorigenicity in mice. To examine the extent of heterogeneity in drug sensitivity, we designed and screened a 200-small molecule compound library in 24 SCPs and the parental cell line. We observed considerable heterogeneity in drug sensitivity and selected 36 compounds for validation, including GPX4 inhibitor ML-210 and gemcitabine, to which the SCPs displayed 12-fold and 150-fold differences in the EC50, respectively. The results of these validation experiments were used to identify transcriptional programs and individual genes which expression correlates with drug sensitivity. We have generated and will evaluate predictive models for drug combination using the SCP drug sensitivity profiles. To test the effect of individual drugs and combinations, as well as different treatment schedules on heterogeneous populations, we will examine the dynamics of individual SCPs within mixed cultures. To this end, we have transduced 22 SCPs with CloneTracer lentiviruses, each encoding for a unique DNA barcode. The distribution of these unique barcodes can be determined by next generation sequencing. Previously, we have found that propagation of a 22 SCP mixture results in reproducible changes in the composition of mixed monolayer cultures with approximately half of the SCPs having a relative abundance of 1% each in the mixed population following propagation for four months. The results of this study show that there is considerable heterogeneity in drug sensitivity among clonal subpopulations derived from a single cancer cell line. Ongoing experiments will provide insight into the effect of drug combinations and unique treatment schedules on distinct tumor cell subpopulations within heterogenous mixtures. Moreover, using this panel of SCPs, we will be able to test for and investigate non-cell autonomous modes of drug resistance, which may guide the development of new drug combinations. Citation Format: Hendrik J. Kuiken, Chandler M. Friend, Steven M. Corsello, Christopher C. Mader, Joan S. Brugge. Characterization of intratumoral heterogeneity in drug sensitivity and modeling of drug combination effects using subclonal cell populations derived from a single breast cancer cell line [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3775.

The nuclear factor κB (NF-κB) signalling pathway controls important cellular events such as cell proliferation, differentiation, apoptosis and immune responses. Pathway activation occurs rapidly upon TNFα stimulation and is highly dependent on ubiquitination events. Using cytoplasmic to nuclear translocation of the NF-κB transcription factor family member p65 as a read-out, we screened a synthetic siRNA library targeting enzymes involved in ubiquitin conjugation and de-conjugation for modifiers of regulatory ubiquitination events in NF-κB signalling. We identified F-box protein only 7 (FBXO7), a component of Skp, Cullin, F-box (SCF)-ubiquitin ligase complexes, as a negative regulator of NF-κB signalling. F-box protein only 7 binds to, and mediates ubiquitin conjugation to cIAP1 and TRAF2, resulting in decreased RIP1 ubiquitination and lowered NF-κB signalling activity.

Growth hormone receptor (GHR) endocytosis is a highly regulated process that depends on the binding and activity of the multimeric ubiquitin ligase, SCF(βTrCP) (Skp Cullin F-box). Despite a specific interaction between β-transducin repeat-containing protein (βTrCP) and the GHR, and a strict requirement for ubiquitination activity, the receptor is not an obligatory target for SCF(βTrCP)-directed Lys(48) polyubiquitination. We now show that also Lys(63)-linked ubiquitin chain formation is required for GHR endocytosis. We identified both the ubiquitin-conjugating enzyme Ubc13 and the ubiquitin ligase COOH terminus of Hsp70 interacting protein (CHIP) as being connected to this process. Ubc13 activity and its interaction with CHIP precede endocytosis of GHR. In addition to βTrCP, CHIP interacts specifically with the cytosolic tails of the dimeric GHR, identifying both Ubc13 and CHIP as novel factors in the regulation of cell surface availability of GHR.

In cancer research the quest continues to identify the Achilles’ heel of cancer. The ideal cancer drug targets are those that are essential in tumor cells but not in normal cells. Such targets are defined as cancer-specific vulnerabilities or as synthetic lethal interactions with cancer-specific genetic lesions. The search for synthetic lethal interactions focuses on proteins that are frequently mutated but elude pharmacological inhibition, for example, RAS, or proteins that are lost in cancer cells and by definition cannot be targeted, such as the tumor suppressor genes p53, APC and RB. These genetic interactions could yield alternative, effective targets for cancer treatment. However, it remains very difficult to predict or extrapolate these synthetic lethal interactions based on existing knowledge. With the discovery of RNAi, unbiased large-scale functional genomic screens for the identification of such targets have become possible potentially leading to major advances in the treatment of cancers. In this review we will discuss the biological basis of synthetic lethal interactions in relation to existing targeted therapeutics, lessons taught by targeted therapeutics already used in the clinic and the implementation of RNAi as tool to identify such synthetic lethal interactions.

Summary Monoubiquitination of FANCD2 and PCNA promotes DNA repair. It causes chromatin accumulation of FANCD2 and facilitates PCNA's recruitment of translesion polymerases to stalled replication. USP1, a protease that removes monoubiquitin from FANCD2 and PCNA, was thought to reverse the DNA damage response of these substrates. We disrupted USP1 in chicken cells to dissect its role in a stable genetic system. USP1 ablation increases FANCD2 and PCNA monoubiquitination but unexpectedly results in DNA crosslinker sensitivity. This defective DNA repair is associated with constitutively chromatin-bound, monoubiquitinated FANCD2. In contrast, persistent PCNA monoubiquitination has negligible impact on DNA repair or mutagenesis. USP1 was previously shown to autocleave after DNA damage. In DT40, USP1 autocleavage is not stimulated by DNA damage, and expressing a noncleavable mutant in the USP1 knockout strain partially rescues crosslinker sensitivity. We conclude that efficient DNA crosslink repair requires FANCD2 deubiquitination, whereas FANCD2 monoubiquitination is not dependent on USP1 autocleavage.

Tumors evolve through progressive accumulation of (epi)genetic alterations, favoring expansion of the fittest cell populations as a result of stimuli and selective pressures in the microenvironment. Intratumor heterogeneity and the interplay of tumor cells with the microenvironment present an order of complexity that can have profound effects on tumor progression and drug sensitivity. While tumor cell heterogeneity has been described at many levels, there is a poor understanding of the extent of functional heterogeneity within a single tumor, and the dynamics and spatial organization of distinct cell populations over time. The overall objective of this study is to characterize the functional heterogeneity of breast tumor cells and evaluate the dynamics of clonal populations within mixtures during tumor progression. We have generated and characterized 31 clonal cell populations (SCP) from the triple-negative breast cancer cell line MDA-MB-468. These SCPs display considerable phenotypic heterogeneity with respect to morphology, proliferation rate, survival in suspension, colony formation in soft agar, and tumorigenicity. We have also performed whole-exome sequencing for 7 SCPs, and RNA-seq and RPPA analysis for all SCPs and the parental cell line. These analyses revealed SCP-unique single nucleotide variants, and differential expression of numerous genes. To study the dynamics of SCP mixtures during the development of xenograft tumors, we have transduced 22 SCPs with unique DNA barcodes derived from the ClonTracer DNA barcode library. The 22 barcoded SCPs were mixed in equal proportions and subsequently injected into the mammary fat pad of NOD/SCID mice. Tumors and lungs were collected at different time points (3 weeks, 2 months and 4 months) to determine the relative changes in clonal representation during tumor progression by next generation sequencing. We identified highly reproducible patterns of clonal expansion, with SCP01 and SCP03 being temporarily enriched for in tumor samples collected at 3 weeks and 2 months respectively, and SCP32 becoming progressively enriched for in tumors over time. In addition, we found recurrent enrichment for SCP01 in most lung samples. Moreover, in contrast to the tumor samples, we found a slight enrichment for SCP13, but not SCP32, during in vitro propagation of the SCP mixture. Together these results suggest the existence of distinct competitive advantages of individual clonal populations within certain spatial and temporal windows. In addition, the discordance in SCP dynamics between the in vivo and in vitro arms suggests that enrichment for SCP01, SCP03 and SCP32 is the result of stimuli or selective pressures that are specific to the tumor microenvironment. Guided by the RNA-seq and RPPA analyses, we are currently testing several hypotheses that may explain the observed enrichment patterns. Citation Format: Hendrik J. Kuiken, Sabin Dhakal, Laura M. Selfors, Jett P. Crowdis, Hyo-eun C. Bhang, Frank Stegmeier, Gordon B. Mills, Joan S. Brugge. Characterization of functional heterogeneity and in vivo dynamics of clonal cell populations derived from the triple-negative breast cancer cell line MDA-MB-468 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3957. doi:10.1158/1538-7445.AM2017-3957

Cisplatin is a widely used chemotherapeutic agent to treat a variety of solid tumors. The cytotoxic mode of action of cisplatin is mediated by inducing conformational changes in DNA including intra- and inter-strand crosslink adducts. Recognition of these adducts results in the activation of the DNA damage response resulting in cell cycle arrest, repair, and potentially, apoptosis. Despite the clinical efficacy of cisplatin, many tumors are either intrinsically resistant or acquire resistance during treatment. The identification of cisplatin drug response modulators can help us understand these resistance mechanisms, provide biomarkers for treatment strategies, or provide drug targets for combination therapy. Here we discuss functional genetic screens, including one performed by us, set up to identify genes whose inhibition results in increased sensitivity to cisplatin. In summary, the validated genes identified in these screens mainly operate in DNA damage response including nucleotide excision repair, translesion synthesis, and homologous recombination.

The transforming growth factor β (TGFβ) pathway orchestrates an extensive transcriptional program that is important for many processes in the cell. For example, TGFβ regulates cell cycle, migration, and epithelial-to-mesenchymal transition. The TGFβ pathway has a dual role in cancer: it is involved in early-stage tumor suppression but also contributes to tumor progression by promoting invasion. To identify the novel genes involved in TGFβ pathway signaling, we have performed a functional genetic loss-of-function screen. We screened a small interfering RNA library targeting 700 kinases and kinase-related genes in a TGFβ-responsive reporter assay. Several genes were identified that upon knockdown could repress the reporter signal; among these are the two cellular receptors for TGFβ. In addition to these two known components of the TGFβ pathway, several genes were identified that were previously not linked to the TGFβ signaling. Knockdown of one of these genes, the IRAK2 kinase, resulted not only in an impaired TGFβ target gene response but also in a reduction of the nuclear accumulation and phosphorylation of SMAD2. In addition, suppression of interleukin-1R–associated kinase 2 expression led to a partial override of a TGFβ-induced cell cycle arrest. Our data show that interleukin-1R–associated kinase 2 is a novel and critical component of TGFβ signaling. Mol Cancer Res; 8(4); 592–603. ©2010 AACR.