The immune checkpoint protein B7H4 is overexpressed in tumors compared to normal tissues, making it an attractive target for cancer immunotherapy. Here, we compare two B7H4-targeting CD3 bispecific antibodies (bsAbs) with different CD3 affinities across in vitro and in vivo characterization studies. In vitro, the CD3xB7H4 bsAb variant with higher CD3 affinity showed greater potency for tumor cell killing and cytokine secretion than the lower affinity variant. In nonclinical toxicology assessments in cynomolgus monkeys, the CD3xB7H4 bsAb variant with lower CD3 affinity induced less cytokine secretion and was tolerated at a higher maximum plasma concentration. Both CD3xB7H4 bsAbs demonstrated antitumor activity in vivo in an ovarian cancer patient-derived xenograft (PDX) mouse model and ex vivo in dissociated primary human ovarian tumor samples. These results demonstrate that increased CD3 affinity enhances cytotoxic potency but results in cytokine secretion at lower antibody concentrations in vitro and in vivo, highlighting an important design trade-off in CD3 bsAb development. The therapeutic potential of CD3xB7H4 bsAbs, particularly in relation to CD3 affinity, warrants further investigation in the clinic.

Objectives: The development of new treatments for hematological cancers faces many challenges and choosing the optimal drug regimen requires careful considerations. Indeed, the various lymphoid malignancies present distinct biological behaviors in terms of tumor proliferation, immune response and escape, and heterogeneous responses to treatment. A QSP platform could be a game-changer by allowing to explore virtually various scenarios and provide valuable insights to clinical research.We developed a mechanistic QSP platform describing different lymphoid malignancies: classical Hodgkin lymphoma (CHL), T-cell and B-cell non-Hodgkin lymphomas (TCL, BCL). Each of these disease models can be coupled with treatment models (pharmacokinetics, mechanism of action). The resulting overall model can be used to explore trial design questions such as optimizing the administration regimen (e.g. dose, frequency).As a use case, we applied this QSP platform to an antibody drug conjugate (ADC) targeting a CDX protein differentially expressed on CHL and TCL cancer cells. We ran in silico clinical trials on virtual populations to identify best treatment regimens in terms of efficacy, and characterize best responders.Methods: We developed a modular QSP platform based on published knowledge and data, implemented as a system of ordinary differential equations. In the ADC use case:- Model was calibrated on public in vitro and human data. and a validation step was performed on PK data.- Simulations and analyses were performed on Jinkō [1]. Results: The final QSP platform integrates: - a mechanistic submodel for each disease of CHL, TCL or BCL, which describes the proliferation of malignant cells, the tumor microenvironment and the intratumor heterogeneity.- a PBPK submodel that predicts the concentration of drugs such as antibodies in the human body.- a submodel accounting for the mechanism of action of the ADC: binding to the target and its internalization, and subsequent release and effect of the payload.The model captures the observed in vitro cytotoxicity, PK dynamics, and human clinical response to treatment. In silico trials, performed in the context of the example use case, allowed to characterize best responders, and optimal dosing regimens in CHL and TCL. It highlighted the importance of both dose and frequency, with higher frequency particularly beneficial at lower doses, for achieving optimal efficacy.Conclusion: This work paves the way towards a QSP platform for blood cancers as a powerful tool to support decision-making in drug development. One key aspect is the ability to progressively integrate a large spectrum of data and knowledge from a diverse set of diseases and treatments. Beyond optimizing trial design and drug regimens, this platform can also provide efficacy estimates for a given lead across multiple indications. Prospective validation of the platform's predictions will be crucial for its integration into drug development pipelines.Citations: [1] https://www.jinko.ai/

Abstract Several clinical trials are currently underway to investigate the effectiveness of CD3 bispecific antibodies (bsAb) in solid tumors. Given the number of approved CD3 bsAb therapies thus far, the development of these bsAbs appears to be more challenging in solid cancer compared to hematological malignancies. The choice of an appropriate tumor-specific target and CD3 affinity are important considerations for the development of effective and safe CD3 bsAbs. The immune checkpoint protein B7H4 is differentially expressed between normal and tumor tissue, showing high expression in various solid cancers. Therefore, B7H4 provides an attractive target for a CD3 bsAb. We explored the preclinical mechanism of action (MoA) and pharmacodynamic (PD) markers of two B7H4-targeting CD3 bsAbs with different CD3 affinities. By crosslinking CD3 on T cells with B7H4 on tumor cells, CD3xB7H4 bsAbs will induce T-cell mediated cytotoxicity in B7H4-expressing tumor cells, associated with T-cell activation and cytokine production. In comparative preclinical characterization studies in vitro, we showed that both bsAbs induced target-specific, dose-dependent, and complete tumor cell kill in a panel of tumor cell lines expressing varying levels of B7H4, albeit with different potency. Moreover, both molecules demonstrated an acceptable safety profile in cynomolgus monkeys. DuoBody-CD3xB7H4 (GEN1047), the variant with lower CD3 affinity, was selected as clinical candidate, to leverage its observed propensity for lower cytokine production. In a patient-derived xenograft model of ovarian cancer in NCG-HIS mice examined in vivo, DuoBody-CD3xB7H4 showed dose-dependent antitumor activity, which was associated with increased numbers of intratumoral T cells and with peripheral PD biomarkers of T-cell activation and cytokine production. Currently, DuoBody-CD3xB7H4 is being investigated in a first-in-human clinical trial for the treatment of solid tumors known to express B7H4 (NCT05180474), in which the MoA and PD, including biomarkers of response, will be clinically explored. Citation Format: Louise A Koopman, Farshid Alemdehy, Madelon Paauwe, Laura Smits-de Vries, Frosso Karaiskaki, Marcel Brandhorst, Patrick Franken, Theo S Plantinga, Mischa A Houtkamp, Stefanie A.H. de Poot, Esther C.W. Breij. Preclinical development of DuoBody®-CD3xB7H4, a novel CD3 bispecific antibody for the treatment of solid cancers [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr IA004.

CD3 bispecific antibodies (bsAbs) show great promise as anticancer therapeutics. Here, we show in-depth mechanistic studies of a CD3 bsAb in solid cancer, using DuoBody-CD3x5T4. Cross-linking T cells with tumor cells expressing the oncofetal antigen 5T4 was required to induce cytotoxicity. Naive and memory CD4+ and CD8+ T cells were equally effective at mediating cytotoxicity, and DuoBody-CD3x5T4 induced partial differentiation of naive T-cell subsets into memory-like cells. Tumor cell kill was associated with T-cell activation, proliferation, and production of cytokines, granzyme B, and perforin. Genetic knockout of FAS or IFNGR1 in 5T4+ tumor cells abrogated tumor cell kill. In the presence of 5T4+ tumor cells, bystander kill of 5T4- but not of 5T4-IFNGR1- tumor cells was observed. In humanized xenograft models, DuoBody-CD3x5T4 antitumor activity was associated with intratumoral and peripheral blood T-cell activation. Lastly, in dissociated patient-derived tumor samples, DuoBody-CD3x5T4 activated tumor-infiltrating lymphocytes and induced tumor-cell cytotoxicity, even when most tumor-infiltrating lymphocytes expressed PD-1. These data provide an in-depth view on the mechanism of action of a CD3 bsAb in preclinical models of solid cancer.

Targeted therapies and immunotherapy have shown promise in patients with non-small cell lung cancer (NSCLC). However, the majority of patients fail or become resistant to treatment, emphasizing the need for novel treatments. In this study, we confirm the prognostic value of levels of AXL, a member of the TAM receptor tyrosine kinase family, in NSCLC and demonstrate potent antitumor activity of the AXL-targeting antibody-drug conjugate enapotamab vedotin across different NSCLC subtypes in a mouse clinical trial of human NSCLC. Tumor regression or stasis was observed in 17/61 (28%) of the patient-derived xenograft (PDX) models and was associated with AXL mRNA expression levels. Significant single-agent activity of enapotamab vedotin was validated in vivo in 9 of 10 AXL-expressing NSCLC xenograft models. In a panel of EGFR-mutant NSCLC cell lines rendered resistant to EGFR inhibitors in vitro, we observed de novo or increased AXL protein expression concomitant with enapotamab vedotin-mediated cytotoxicity. Enapotamab vedotin also showed antitumor activity in vivo in 3 EGFR-mutant, EGFR inhibitor-resistant PDX models, including an osimertinib-resistant NSCLC PDX model. In summary, enapotamab vedotin has promising therapeutic potential in NSCLC. The safety and preliminary efficacy of enapotamab vedotin are currently being evaluated in the clinic across multiple solid tumor types, including NSCLC.

Intratumor heterogeneity is a key factor contributing to therapeutic failure and, hence, cancer lethality. Heterogeneous tumors show partial therapy responses, allowing for the emergence of drug-resistant clones that often express high levels of the receptor tyrosine kinase AXL. In melanoma, AXL-high cells are resistant to MAPK pathway inhibitors, whereas AXL-low cells are sensitive to these inhibitors, rationalizing a differential therapeutic approach. We developed an antibody-drug conjugate, AXL-107-MMAE, comprising a human AXL antibody linked to the microtubule-disrupting agent monomethyl auristatin E. We found that AXL-107-MMAE, as a single agent, displayed potent in vivo anti-tumor activity in patient-derived xenografts, including melanoma, lung, pancreas and cervical cancer. By eliminating distinct populations in heterogeneous melanoma cell pools, AXL-107-MMAE and MAPK pathway inhibitors cooperatively inhibited tumor growth. Furthermore, by inducing AXL transcription, BRAF/MEK inhibitors potentiated the efficacy of AXL-107-MMAE. These findings provide proof of concept for the premise that rationalized combinatorial targeting of distinct populations in heterogeneous tumors may improve therapeutic effect, and merit clinical validation of AXL-107-MMAE in both treatment-naive and drug-resistant cancers in mono- or combination therapy.

Abstract Enhanced AXL expression has been observed in tumor tissues obtained from non-small cell lung (NSCLC) patients, both in drug-treatment naïve patients and in patients with acquired resistance to EGFR tyrosine kinase inhibitors (EGFRi). In this study, we have evaluated the antitumor activity of the novel AXL-targeting antibody-drug conjugate (ADC) AXL-107-MMAE (HuMax-AXL-ADC) in non-small cell lung cancer (NSCLC) in vitro and in vivo. The antitumor activity of AXL-107-MMAE (4 mg/kg) was evaluated in vivo in a mouse patient-derived xenograft (PDX) clinical trial (1 mouse per group), using a collection of 57 NSCLC-derived PDX models, encompassing the typical NSCLC histological subtypes and mutational genotypes. AXL-107-MMAE induced responses, defined by a decrease in relative tumor growth compared to an untreated control tumor, in 35/57 (61%) of PDX models. Potent anti-tumor activity, i.e. tumor stasis or tumor regression, was observed in 16 out of 57 (28%) models. Potent anti-tumor activity was associated with higher AXL RNA expression compared to models showing intermediate response or non-responders (p<0.001). The therapeutic activity of AXL-107-MMAE (2 and 4 mg/kg) was confirmed in a panel of NSCLC PDX or cell line-derived xenograft models using 6-8 mice per group. AXL-107-MMAE induced dose-dependent, single agent anti-tumor activity in 8 out of 9 models. These included 2 EGFR-mutant PDX models that were resistant to the EGFR inhibitor erlotinib, including one model containing L858R/T790M EGFR mutations and one model with a L858R EGFR mutation. To specifically evaluate opportunities for AXL-107-MMAE in EGFR-mutant, treatment-resistant NSCLC, we established a panel of EGFR-mutant NSCLC cell lines with acquired resistance to the EGFRi erlotinib, gefitinib, or osimertinib. In general, cell lines showed enhanced AXL protein expression upon acquiring resistance to EGFRi. Moreover, the AXL-expressing, EGFRi-resistant cell lines were efficiently killed by AXL-107-MMAE in vitro, whereas the parental, EGFR-TKI sensitive NSCLC cell lines, which expressed little or no AXL, did not respond to treatment with AXL-107-MMAE. In summary, we show that AXL-107-MMAE has therapeutic activity as a single agent in the majority of NSCLC-derived models evaluated in vivo, representing different NSCLC histological and mutational subtypes. Moreover, AXL-107-MMAE induced efficient cytotoxicity in NSCLC cell lines that showed enhanced AXL expression upon acquired resistance to EGFRi. Finally, AXL-107-MMAE induces anti-tumor activity in EGFR-mutant, EGFRi resistant NSCLC PDX models. Citation Format: Louise A. Koopman, Maarten L. Janmaat, Kirstine Jacobsen, Mikkel Green Terp, Elke Gresnigt-van den Heuvel, Ulf Forssman, Andreas Lingnau, Paul W. Parren, Henrik Ditzel, Esther C. Breij. An AXL-specific antibody-drug conjugate shows preclinical anti-tumor activity in non-small cell lung cancer, including EGFR-inhibitor resistant NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 832.

BACKGROUND:Chemotherapeutic efficacy can be improved by targeting the structure and function of the extracellular matrix (ECM) in the carcinomal stroma. This can be accomplished by e.g. inhibiting TGF-β1 and -β3 or treating with Imatinib, which results in scarcer collagen fibril structure in xenografted human KAT-4/HT29 (KAT-4) colon adenocarcinoma.METHODS:The potential role of αVβ6 integrin-mediated activation of latent TGF-β was studied in cultured KAT-4 and Capan-2 human ductal pancreatic carcinoma cells as well as in xenograft carcinoma generated by these cells. The monoclonal αVβ6 integrin-specific monoclonal antibody 3G9 was used to inhibit the αVβ6 integrin activity.RESULTS:Both KAT-4 and Capan-2 cells expressed the αVβ6 integrin but only KAT-4 cells could utilize this integrin to activate latent TGF-β in vitro. Only when Capan-2 cells were co-cultured with human F99 fibroblasts was the integrin activation mechanism triggered, suggesting a more complex, fibroblast-dependent, activation pathway. In nude mice, a 10-day treatment with 3G9 reduced collagen fibril thickness and interstitial fluid pressure in KAT-4 but not in the more desmoplastic Capan-2 tumors that, to achieve a similar effect, required a prolonged 3G9 treatment. In contrast, a 10-day direct inhibition of TGF-β1 and -β3 reduced collagen fibril thickness in both tumor models.CONCLUSION:Our data demonstrate that the αVβ6-directed activation of latent TGF-β plays a pivotal role in modulating the stromal collagen network in carcinoma, but that the sensitivity to αVβ6 inhibition depends on the simultaneous presence of alternative paths for latent TGF-β activation and the extent of desmoplasia.

Abstract Upon therapeutic pressure, cancers commonly select for drug-resistant, invasive subpopulations with elevated expression of the receptor tyrosine kinase AXL. Besides the correlation between high AXL expression and induction of epithelial-to-mesenchymal transition, a process known to support metastasis, ample evidence also links AXL to resistance against a variety of targeted therapies, including inhibitors of the MAPK pathway in malignant melanoma and the EGFR pathway in lung cancer. AXL-107-MMAE (HuMax-AXL-ADC) is a therapeutic antibody-drug conjugate specific for AXL, containing the microtubule disrupting agent monomethyl auristatin E as the cytotoxic payload. AXL-107-MMAE was previously shown to induce potent cytotoxicity in vitro and in vivo, which was dependent expression of AXL on the cell surface. We evaluated the capacity of AXL-107-MMAE to target AXL-positive MAPK pathway inhibitor resistant tumor cells, using malignant melanoma as a clinically relevant example. First, AXL-107-MMAE was shown to induce cytotoxicity in BRAF-mutant tumor cell lines that showed AXL expression upon acquired resistance to BRAF-inhibitors. In contrast, no cytotoxicity was observed in the BRAF-inhibitor-sensitive, AXL-negative parental cell lines. In heterogeneous tumor cell cultures, treatment with a BRAF-inhibitor selected for AXL-high, MAPK pathway inhibitor-insensitive melanoma cells, which was prevented by combined BRAF-inhibitor and AXL-107-MMAE treatment. Interestingly, we observed marked AXL upregulation in biopsies obtained from patients after they developed resistance to MAPK pathway inhibitors compared to paired pre-treatment biopsies. The in vivo potential of AXL-107-MMAE in malignant melanoma was demonstrated using BRAFV600E-mutated xenograft model derived from a patient who developed resistance to the BRAF inhibitor vemurafenib in the clinic. This indicates that AXL expression levels in treatment-resistant malignant melanoma are sufficient to induce tumor regression with AXL-107-MMAE, at least in this model system. These findings merit clinical validation of the targeting of both treatment-naïve and drug-resistant cancers with AXL-107-MMAE, either alone or in combination with other targeted therapies. Citation Format: Julia Boshuizen, Louise A. Koopman, Esther C. Breij, David Satijn, Daniel Peeper, Paul W. Parren. Specific elimination of invasive and multidrug-resistant cancer cells by an antibody-drug conjugate targeting AXL [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 4591. doi:10.1158/1538-7445.AM2017-4591

La presente divulgation concerne des conjugues anticorps-medicament (ADC) se liant a l'AXL humain a usage therapeutique, en particulier pour le traitement des cancers resistants ou refractaires.

Tyro3, a member of the Tyro3/Axl/Mer (TAM) family of receptor tyrosine kinases, has emerged as a potential oncogene in melanoma. Here, we confirm that Tyro3 is specifically overexpressed in primary melanoma samples and show that Tyro3 is expressed at varying levels in numerous melanoma cell lines. Short hairpin RNA-mediated knockdown of Tyro3 led to significant cell death via apoptotic mechanisms in nearly all melanoma cell lines tested, regardless of the BRAF or NRAS mutation status or co-expression of Axl and/or Mer. We generated soluble and monomeric versions of the human Tyro3 extracellular domain and human Gas6 for affinity measurements and correlated these values with the level of Gas6 required to induce Tyro3 signaling in cellular assays. Calcium was critical for the correct folding of Gas6 and its binding to Tyro3. In melanoma cell lines, Gas6 induced Tyro3 phosphorylation and downstream Akt phosphorylation without apparent effects on Erk. We generated monoclonal antibodies (mAbs) against Tyro3 to examine their effect on survival signaling in melanoma cell lines. The mAbs generated against Tyro3 included nonligand blockers, partial blockers, and competitive ligand blockers. A number of weak and partial ligand blockers (all recognizing the Tyro3 Ig domains) were the most effective at blocking ligand-mediated downstream signaling of Tyro3. Overall, these data indicate that Tyro3 may confer increased survival signals in melanoma cells and can be stymied using inhibitory mAbs. These mAbs may be useful for further investigations of the role of Tyro3 in melanoma.

First trimester human decidua is composed of decidual cells, CD56(bright)CD16(-) decidual natural killer (dNK) cells, and macrophages. Decidual cells incubated with NK cell-derived IFN-gamma and either macrophage-derived TNF-alpha or IL-1 beta synergistically enhanced mRNA and protein expression of IP-10 and I-TAC. Both chemokines recruit CXCR3-expressing NK cells. This synergy required IFN-gamma receptor 1 and 2 mediation via JAK/STAT and NF kappa B signaling pathways. However, synergy was not observed on neutrophil, monocyte, and NK cell-recruiting chemokines. Immunostaining of first trimester decidua localized IP-10, I-TAC, IFN-gamma R1, and -R2 to vimentin-positive decidual cells versus cytokeratin-positive interstitial trophoblasts. Flow cytometry identified high CXCR3 levels on dNK cells and minority peripheral CD56(bright)CD16(-) pNK cells and intermediate CXCR3 Levels on the majority of CD56(dim)CD16(+) pNK cells. Incubation of pNK cells with either IP-10 or I-TAC elicited concentration-dependent enhanced CXCR3 Levels and migration of both pNK cell subsets that peaked at 10 ng/mL, whereas each chemokine at a concentration of 50 ng/mL inhibited CXCR3 expression and pNK cell migration. Deciduae from women with preeclampsia, a leading cause of maternal and fetal morbidity and mortality, displayed significantly lower dNK cell numbers and higher IP-10 and I-TAC levels versus gestational age-matched controls. Significantly elevated IP-10 levels in first trimester sera from women eventually developing preeclampsia compared with controls, identifying IP-10 as a novel, robust early predictor of preeclampsia.

First trimester human decidua is composed of decidual cells, CD56(bright)CD16(-) decidual natural killer (dNK) cells, and macrophages. Decidual cells incubated with NK cell-derived IFN-γ and either macrophage-derived TNF-α or IL-1β synergistically enhanced mRNA and protein expression of IP-10 and I-TAC. Both chemokines recruit CXCR3-expressing NK cells. This synergy required IFN-γ receptor 1 and 2 mediation via JAK/STAT and NFκB signaling pathways. However, synergy was not observed on neutrophil, monocyte, and NK cell-recruiting chemokines. Immunostaining of first trimester decidua localized IP-10, I-TAC, IFN-γR1, and -R2 to vimentin-positive decidual cells versus cytokeratin-positive interstitial trophoblasts. Flow cytometry identified high CXCR3 levels on dNK cells and minority peripheral CD56(bright)CD16(-) pNK cells and intermediate CXCR3 levels on the majority of CD56(dim)CD16(+) pNK cells. Incubation of pNK cells with either IP-10 or I-TAC elicited concentration-dependent enhanced CXCR3 levels and migration of both pNK cell subsets that peaked at 10 ng/mL, whereas each chemokine at a concentration of 50 ng/mL inhibited CXCR3 expression and pNK cell migration. Deciduae from women with preeclampsia, a leading cause of maternal and fetal morbidity and mortality, displayed significantly lower dNK cell numbers and higher IP-10 and I-TAC levels versus gestational age-matched controls. Significantly elevated IP-10 levels in first trimester sera from women eventually developing preeclampsia compared with controls, identifying IP-10 as a novel, robust early predictor of preeclampsia.

Mutational K-Ras activation arises very frequently in epithelial cancers. However, the specific role of oncogenic K-Ras in maintaining tumor cell survival is unclear. To analyze K-Ras dependency in human cancers we have employed K-Ras-specific shRNAs to deplete K-Ras protein in cancer cell lines derived from lung and pancreatic adenocarcinomas harboring oncogenic K-Ras mutations. By this approach two classes of cell lines were identified - those that are addicted to or dependent on K-Ras activity for viability and those that are independent. We have identified a number of cellular and molecular features that can distinguish these two classes. Firstly, K-Ras dependent cells are epithelial whereas independent cell lines are mesenchymal, as assessed by E-Cadherin and vimentin expression. Secondly, K-Ras dependent cells exhibit high-level focal amplification of the K-Ras gene resulting in increased K-Ras gene copy number and overexpression of mutant K-Ras protein. This phenomenon is reminiscent of addiction to EGFR in lung cancers. Lastly, by comparing gene expression profiles for \#8220;K-Ras-dependent\#8221; and \#8220;K-Ras independent\#8221; cancer cells, we established a gene expression signature that accurately predicts K-Ras dependency and is associated with the differentiation state or histological grade of human lung tumors. We have identified three genes differentially expressed in K-Ras dependent cells that could represent pharmacologically tractable therapeutic targets. These are ITGB6, encoding the integrin beta-6 subunit, as well as SYK and MST1R, which encode tyrosine kinases. Disruption of these genes by RNAi induces epithelial-mesenchymal transformation and apoptosis specifically in K-Ras-dependent cells. Therefore, oncogenic \#8220;K-Ras addiction\#8221; and epithelial differentiation are associated and we suggest that genes regulating epithelial plasticity in K-Ras-addicted cancers may represent novel therapeutic targets. Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 1882.

K-ras mutations occur frequently in epithelial cancers. Using short hairpin RNAs to deplete K-Ras in lung and pancreatic cancer cell lines harboring K-ras mutations, two classes were identified-lines that do or do not require K-Ras to maintain viability. Comparing these two classes of cancer cells revealed a gene expression signature in K-Ras-dependent cells, associated with a well-differentiated epithelial phenotype, which was also seen in primary tumors. Several of these genes encode pharmacologically tractable proteins, such as Syk and Ron kinases and integrin beta6, depletion of which induces epithelial-mesenchymal transformation (EMT) and apoptosis specifically in K-Ras-dependent cells. These findings indicate that epithelial differentiation and tumor cell viability are associated, and that EMT regulators in "K-Ras-addicted" cancers represent candidate therapeutic targets.