MALT1 is a key component of the CARD11-BCL10-MALT1 (CBM) complex downstream from BTK on the B-cell receptor signaling pathway. It is a key mediator of NF-κB signaling and considered a potential therapeutic target for several subtypes of non-Hodgkin's B-cell lymphomas. By applying advanced physics-based modeling techniques, including combining free energy calculations with machine learning methods and a chemistry-aware compound enumeration workflow, extensive sets of de novo design ideas were explored to quickly identify a novel hit series. Multiparameter optimization allowed efficient prioritization of molecules with good potency and drug-like properties during lead optimization, which led to the discovery of a highly potent MALT1 inhibitor, SGR-1505, with a well-balanced property profile. It demonstrated strong antitumor activity alone and in combination with BTK inhibitor in multiple in vivo B-cell lymphoma xenograft models and progressed to a phase 1 clinical trial in patients with mature B-cell neoplasms.

Background: MALT1, a core subunit of the CBM complex (CARD11-BCL10-MALT1), plays an essential role in the NF-kB pathway activation downstream of BTK in the B-cell receptor signaling pathway, promoting growth and survival of B-cell malignancies. SGR-1505, a potent and selective MALT1 inhibitor, demonstrates monotherapy and combination activity in B-cell lymphoma models. Translation of single agent clinical activity has been demonstrated in patients with R/R B-cell malignancies, including WM with prior BTKi therapy and CLL/SLL with ≥2 prior lines of therapy (Spurgeon et al. EHA 2025). Initial safety and efficacy data are updated, and pathogenic gene mutations of interest from responding patients are described. Aim: To evaluate the activity of SGR-1505 in overcoming BTK resistance in B-cell malignancies as a single agent or in combination with a BCL-2 inhibitor or BTK degrader preclinically. To characterize the mutational profile and clinical response to SGR-1505 treatment in patients with B-cell malignancies who have relapsed or become refractory to BTKi treatment or who have been double-exposed to BTKi and BCL2i. Methods: SGR-1505 anti-proliferative activity alone or in combination with venetoclax or BTK degrader NX-2127 were evaluated in BTKi-resistant cell lines in vitro and in vivo. OCI-LY3 cell line, a cell line derived from ABC-DLBCL, is primarily resistant to BTKi due to its CARD11 L251P mutation. Cell lines with acquired resistance to BTKi were generated by continuous passage with BTK inhibitors or by introducing BTK C481S or BTK L528W through CRISPR-Cas9 gene editing. Monotherapy clinical activity of SGR-1505 is being evaluated in a phase-1 dose-escalation study in patients with relapsed or refractory B-cell malignancies (NCT05544019). Samples for biomarker analysis were collected at baseline and at scheduled visits. Mutational status of BTK, BCL2 and other genes of interest were taken from medical history, profiled in ctDNA or in biopsies when available. Results: SGR-1505 blocked the caspase mediated cleavage of NF-kB regulators, activation of the downstream NF-kB pathway, and inhibited LY3 cell growth in vitro and in vivo. In B-cell lymphoma lines with acquired BTKi resistance, SGR-1505 exhibited comparable IC50 values as compared to the parental cell lines, and combining SGR-1505 with venetoclax or NX-2127 enhanced growth inhibition. As of 13 May 2025, a total of 49 patients had been treated in study SGR-1505-101, including 6 WM relapsing or refractory to BTKi and 6 CLL/SLL who were double-exposed to prior BTKi and BCL2i. In the 6 WM subjects, median prior lines of therapy was 2 (range: 2-6) and all received prior BTKi as their last line of therapy and were refractory or relapsed due to development of BTK resistance mutations or bypass mechanisms. Four of the 6 had documented MYD88 mutations, and 1 also carried a CXCR4 mutation. SGR-1505 induced rapid IgM decreases. All 6 WM patients have had objective responses (3 minor responses, 3 partial responses, including 1 PR deepening from prior MR and one additional PR following the 13 May 2025 cut-off-date). In the six “double-exposed” CLL/SLL patients, the median prior lines of therapy was 4 (range: 2-7). Two achieved objective partial response (PR or PR-L), including a patient who received 7 lines of prior therapy and carried BCL2 resistance mutation D103Y. During dose escalation, SGR-1505 has also demonstrated activity in other B-cell malignancies, including a complete response in a subject with ABC-DLBCL (deepening from prior PR, confirmed after the data cut), a PR in a subject with MZL who had previously received BTKi, and a PR in a CLL/SLL subject who relapsed following prior chemotherapy. Mutational profiling of BTKi and BCL2 resistance mutations and other genes of interest in SGR-1505 responders is ongoing. Conclusion: Preclinically, SGR-1505 demonstrated the ability to overcome BTKi resistance in multiple in vitro and in vivo models. Clinically, SGR-1505 demonstrated encouraging anti-tumor activity in prior BTKi exposed WM, BTKi/BCL2i double-exposed CLL/SLL and other B-cell malignancies. MALT1 inhibition represents a promising novel therapeutic option with broad activity across multiple tumor types and the potential to overcome BTK and BCL2 resistance.

Transfer does not induce homeostatic inhibition on CD4+ T-cells

Immunotransplant modulates serum cytokines levels

Immunotransplant requires double checkpoint blockade

Background: MALT1 (Mucosa-associated lymphoid tissue lymphoma translocation protein 1) is a component of the MALT1-BCL10-CARD11 complex downstream from the Bruton Tyrosine Kinase (BTK) on the B-cell receptor signaling pathway. MALT1 is a key mediator of nuclear factor kappa B (NF-κB) signaling, which is the main driver of a subset of B-cell lymphomas. MALT1 is considered a potential therapeutic target for several subtypes of non-Hodgkin B-cell lymphomas and chronic lymphocytic leukemia (CLL), including tumors with acquired BTK inhibitor (BTKi) resistance. Constitutive activation of the NF-κB is a molecular hallmark of activated B cell-like diffuse large B cell lymphoma (ABC-DLBCL), and MALT1 may have utility as a treatment option for ABC-DLBCL. Previously, we described the discovery of novel MALT1 inhibitors with anti-proliferative effects in non-Hodgkin B-cell lymphoma cells and the strong anti-tumor activity of our MALT1 inhibitors across multiple tumor models as well as combination potential with agents including standard-of-care (ref 1, 2). SGR-1505 is an oral potent small molecule allosteric inhibitor of MALT1 that inhibits MALT1 enzymatic activity and demonstrates anti-proliferative activity in ABC-DLBCL cell lines, both BTKi-sensitive (OCI-LY10) and BTKi-resistant (OCI-LY3). When administered as a single agent and in combination with the approved Bruton's tyrosine kinase (BTK) inhibitor, ibrutinib, SGR-1505 demonstrated tumorostatic and regressive antitumor activity in ABC-DLBCL cell line-derived xenograft and patient-derived xenograft models. These data suggest that SGR-1505-mediated MALT1 inhibition has therapeutic potential for patients with selected B-cell lymphomas. Here we further characterized SGR-1505, in a series of in vitro and ex vivo assays, as well as RNA-seq analysis to examine changes in gene expression from in vivo tumor samples. We also compared SGR-1505 with a competitor Phase I candidate, JNJ-67856633 (JNJ-6633) (ref 3, 4). Results: SGR1505 potency and downstream effects were evaluated in a series of biochemical and cell based assays. SGR-1505 showed excellent potency in the biochemical assay and strong anti-proliferative effects on ABC-DLBCL cells. SGR-1505 was more potent than JNJ-6633 in all assays tested (Table 1). These results are also consistent with the result from a human primary T-cell based assay, where SGR-1505 showed at least ten-fold better potency than JNJ-6633. RNA-seq analysis was conducted to examine changes in gene expression from in vivo tumor samples. Greater modulation of BIOCARTA NF-kB pathway genes was seen with SGR-1505 compared to JNJ-6633, as measured by mean absolute change in gene expression. At 6 hr and later timepoints, we also observed a trend of increases in genes related to cell cycle pathways, such as cell cycle, DNA damage, and apoptosis. SGR-1505 is being evaluated in the SGR-1505-102 phase 1 study, which is an ongoing first-in-human, single center, dose escalation study to evaluate the safety, tolerability, PK and PD of SGR-1505 tablets in healthy participants (ACTRN12623000358640p). Preliminary data showed changes in target engagement markers at concentrations predicted by the in vitro and ex vivo assays, consistent with MALT1 protease inhibition. Conclusions: SGR-1505, a MALT1 protease small molecule inhibitor, consistently demonstrated better potency in in vitro and ex vivo assays when compared to the clinical-stage JNJ-6633 compound and greater effects on NF-kB pathway gene expression in in vivo tumor samples based on RNA-seq analysis. Changes in biological pathways mediated by MALT1 were also observed at relevant doses in the ongoing SGR-1505 healthy volunteer study. Currently, a phase 1 clinical trial in patients with mature B cell neoplasms is also ongoing (NCT05544019). The data presented suggests SGR-1505 has a potential best-in-class profile and supports advancing the ongoing clinical development of SGR-1505.

Transfer induces homeostatic inhibition

Homeostatic activation and inhibition require JAK3-signaling

Introduction: MALT1 (mucosa-associated lymphoid tissue lymphoma translocation protein 1) is a key mediator of the NF-κB signaling pathway, the main driver of a subset of B-cell lymphomas and functions by forming a complex with CARMA1 and BCL10 to mediate antigen receptor-induced lymphocyte activation. MALT1 is considered a potential therapeutic target for several subtypes of non-Hodgkin B-cell lymphomas and chronic lymphocytic leukemia (CLL). Previously, we described the discovery of novel and potent MALT1 inhibitors with anti-proliferative effects in non-Hodgkin B-cell lymphoma cells. Here, we highlight the strong anti-tumor activity of our MALT1 inhibitors across multiple tumor models and the combination potential with agents including standard-of-care.

2593 Background: Patients with refractory diffuse large b cell lymphoma (DLBCL) have poor outcomes with < 30% surviving for 12 months and especially poor outcomes for those who progress after chimeric antigen receptor T cells (CAR-T) or autologous stem cell transplant (ASCT). These therapies utilize lymphodepleting chemotherapy which induces homeostatic T cell proliferation. We have demonstrated these expanding T cells express high levels of PD1 and CTLA4. In pre-clinical models, addition of dual checkpoint blockade (DCB) with anti-PD1/anti-CTLA-4 to adoptive T cell transfer after lymphodepletion achieved a synergistic anti-tumor effect. Based on these studies, we developed a phase Ib/II study of Nivolumab/Ipilimumab primed “immunotransplant” for relapsed/refractory (R/R) DLBCL (NCT03305445). Methods: Phase Ib of the trial enrolled 6 patients with progressive disease following at least one line of standard therapy. Patients received two cycles of DCB with ipilimumab (1mg/kg) and nivolumab (3mg/kg) given at three-week intervals followed by immunotransplant (i.e. peripheral blood T cell harvest, lymphodepletion with fludarabine/cyclophosphamide, T cell reinfusion), followed by two further cycles of DCB and nivolumab maintenance. Results: Five patients received at least two cycles of DCB and the autologous T cell transfer, while one patient had progressive disease during initial DCB and required salvage chemotherapy. Treatment emergent AE (TEAE) occurred in 100% of patients. As expected with lymphodepleting chemotherapy, the most common TEAE were neutropenia (66.7% grade 1, 66.7% grade ≥ 3), fatigue (83.3%, 16.7%), fever (66.7%, 0%), and dyspnea (66.7%, 0.0%). One patient (16.7%) died during the intervention period (grade 5 TEAE), though relation to study drug is unclear. Three patients (50.0%) experienced clinical benefit with immunotransplant. One patient (a 58yo M with progression after seven lines of therapy including ASCT and CAR-T) is experiencing partial metabolic response after 4 months on protocol. One 77yo F with multiple prior lines of therapy including ASCT has experienced an extended complete metabolic response, currently 31 months post immunotransplant. A third (a 50yo F) experienced mixed radiographic response, and has not received subsequent therapy 30 months following immunotransplant. Conclusions: Nivolumab/Ipilimumab primed immunotransplant is well tolerated in patients with R/R DLBCL for whom there are few treatment options. Preliminary results demonstrate remissions in heavily pre-treated patients, including prior ASCT and CAR-T. Pre-clinical models in melanoma, non-small cell lung cancer, and T cell lymphoma all demonstrate synergy when DCB is administered with lymphodepletion and autologous T cell transfer. These data support further investigation of DCB-primed immunotransplant. Clinical trial information: NCT03305445.

Abstract T-cell transfer into lymphodepleted recipients induces homeostatic activation and potentiates antitumor efficacy. In contrast to canonical T-cell receptor–induced activation, homeostatic activation yields a distinct phenotype and memory state whose regulatory mechanisms are poorly understood. Here, we show in patients and murine models that, following transfer into lymphodepleted bone marrow transplant (BMT) recipients, CD8+ T cells undergo activation but also simultaneous homeostatic inhibition manifested by upregulation of immune-checkpoint molecules and functional suppression. T cells transferred into BMT recipients were protected from homeostatic inhibition by PD-1/CTLA4 dual checkpoint blockade (dCB). This combination of dCB and BMT—”immunotransplant”—increased T-cell homeostatic activation and antitumor T-cell responses by an order of magnitude. Like homeostatic activation, homeostatic inhibition is IL7/IL15-dependent, revealing mechanistic coupling of these two processes. Marked similarity in ex vivo modulation of post-BMT T cells in mice and patients is promising for the clinical translation of immunotransplant (NCT03305445) and for addressing homeostatic inhibition in T-cell therapies. Significance: For optimal anticancer effect, T-cell therapies including chimeric antigen receptor T-cell, tumor-infiltrating lymphocyte, and transgenic T-cell therapies require transfer into lymphodepleted recipients and homeostatic activation; however, concomitant homeostatic inhibition mitigates T-cell therapies' efficacy. Checkpoint blockade uncouples homeostatic inhibition from activation, amplifying T-cell responses. Conversely, tumors nonresponsive to checkpoint blockade or BMT are treatable with immunotransplant. See related commentary by Ansell, p. 1487. This article is highlighted in the In This Issue feature, p. 1469

123 Background: In patients with low-grade lymphoma, in situ vaccination has yielded both partial and complete remissions in clinical trials. Though clinical responses have been observed with multiple pattern recognition receptor agonists (PRRa), the optimal immune stimulant is unknown. We hypothesize that natural PRRa, such as the attenuated pathogens or subunits found in common prophylactic vaccines, could target multiple PRR in a physiologically relevant context and lead to a more robust activation of dendritic cells (DCs) versus synthetic PRRa. Methods: 20 vaccines, including BCG, Typhim Vi, MMR-II, etc. were screened in vitro, where DC phenotype and function were evaluated by flow cytometry. Flt3L-mobilized DC ability to phagocytose, process, present, and cross-present soluble protein or tumor derived antigen, were assessed using CRISPR gene-edited, β2m(-/-) GFP-lymphoma cells and a novel GFP-specific (‘JEDI’) CD8 T cell system. Vaccine mechanism of immune activation was elucidated using a library of PRR-null macrophage cell lines. Potent vaccines were also evaluated in vivo in a Flt3L-primed in situ vaccination using the A20 murine lymphoma model. Results: Several vaccines induced robust DC activation and several showed significant increases in subsequent T cell activation, proliferation, and tumor killing, suggesting increased antigen processing and cross-presentation by DCs. Some vaccines, either as single agents or in combination, were significantly more effective than synthetic PRRa in activating DCs and inducing a T cell response. In vivo, vaccine combination therapies induced tumor regression in a majority of animals, suggesting synergistic immune activation. Conclusions: This data suggests prophylactic vaccines are effective clinical-grade DC activators and can be repurposed for use in the in situ vaccination maneuver, with immediate translation into the clinic. Additionally, by extensive in vitro evaluation in parallel with in vivo studies, this work aims to identify a predictive in vitro molecular immune signature that correlates closely with adjuvant efficacy in vivo.

Abstract Lymphomas, with an estimated 80,000 new cases, are the 5th most common cancer in the United States and the majority of lymphoma subtypes are incurable and in need of novel, mechanistically distinct therapies. Two approaches to potently increase the anti-tumor potential of T cells are: checkpoint blockade (e.g. anti-CTLA-4 and anti-PD-1 antibodies) and adoptive T-cell transfer into lympho-depleted recipients. We have developed a novel therapy combining these approaches into ‘checkpoint-blockade-primed immunotransplant’ comprised of: -treatment of tumor-bearing host with anti-CTLA-4 and/or anti-PD-1 antibodies -splenocyte harvest and transfer to lympho-depleted recipient Whereas checkpoint blockade alone has minimal anti-tumor effect in the pre-clinical model, our results show that the combined therapy results in superior anti-tumor immunity as seen by increased production of tumor-reactive IFN γ positive T-cells. Treatment of both tumor-bearing donor and recipient with checkpoint antibodies induces cure of the majority of recipients, in a CD8, NK, and IFNγ-dependent manner. Furthermore, we have demonstrated that CD8 T cells exposed to checkpoint blockade and transfer into the lymphopenic host demonstrate greater: -in vivo serum levels of IL-15 and IL-7 -surface expression levels of IL-15R and IL-7R -in vitro STAT5 phosphorylation in response to common γ-chain cytokines -in vivo proliferation in response to exposure to cognate tumor antigen Ongoing studies will seek to assess the dependence of the above observations (cytokine production, proliferation, anti-tumor effect) on specific common γ-chain cytokines, the role of lymphopenia in inducing T cell trafficking to tumor versus healthy tissue, and guide development of the immunotransplant model to optimize the amplification of anti-tumor immunity for near-term clinical translation. Citation Format: Netonia Marshall, Thomas Marron, Judith Agudo, Brian Brown, Joshua Brody. Immunotransplant: Merging checkpoint blockade and T-cell transfer into lymphodepleted recipients [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B107.

### P189 Rational combinations of intratumoral T cell and myeloid agonists mobilize abscopal responses in prostate cancer #### Casey Ager1, Matthew Reilley2, Courtney Nicholas1, Todd Bartkowiak1, Ashvin Jaiswal1, Michael Curran1 ##### 1Department of Immunology, University of Texas MD Anderson

e14538 Background: Lymphomas comprise the 5thmost common cancers in the U.S. and the majority of these are incurable with standard chemo-immunotherapy, thus, novel, mechanistically distinct therapies are needed, such as immunotherapy. Two promising classes of immunotherapy are: checkpoint blockade (e.g. anti-PD1 and anti-CTLA-4 antibody) and adoptive T-cell transfer lymphocytes into lymphodepleted recipients (e.g. CARs and TILs). Methods: In a PDL1(+), CD80/86(+) murine lymphoma model, we have developed a novel therapy combining these approaches into 'checkpoint-blockade-primed immunotransplant' comprised of: -treatment of tumor-bearing animals (donors) with anti-PD-1 and anti-CTLA-4 antibodies -splenocyte and bone marrow harvest and transfer to lymphodepleted/myeloablated (9Gy TBI) recipient Results: The combined therapy immunotransplant maneuver results in superior anti-tumor immunity compared to either checkpoint blockade or syngeneic transplant individually. Transferred T cells significantly increase surface and intra-cellular PD1 and CTLA-4, respectively, in both CD4 and CD8 T cells. Treatment of both tumor-bearing donor and recipient with anti-PD1 and anti-CTLA-4 antibodies induces cure of the majority of recipients, in a CD8 and IFNγ-dependent manner, despite the finding that antibody therapy alone (without transplantation and T cell transfer) induces minimal anti-tumor effect. Herein, we have demonstrated that T cells exposed to checkpoint blockade and transfer into the lymphopenic host demonstrate greater: -response to common γ-chain cytokines (per in vitro STAT5 phosphorylation) -in vitro production of IFNγ and TNF production in response to exposure to cognate tumor antigen -in vivo proliferation after exposure to tumor antigen Conclusions: Overall the data suggest both that: lymphopenia-exposed T cells become activated by checkpoint blockade and checkpoint blockade-exposed T cells become more activated by common γ-chain cytokines associated with lymphopenia. These data guide the development of combination therapies such as immunotransplant which we demonstrate is uniquely capable of curing the majority of established tumors.

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CAHER2 is a receptor tyrosine kinase found overexpressed in 20-40% of breast tumors correlating with a malignant phenotype and worse prognosis. Different targeted therapies have been developed to specifically inhibit its activity such the monoclonal antibodies Trastuzumab and Pertuzumab or the small tyrosine kinase inhibitor Lapatinib. Unfortunately, a large number of these patient tumors eventually progress, acquiring resistance..Our main goal is to identify which pathways have become essential for HER2 overexpressing cells but are not needed for normal cells to survive. To determine this, we performed a pooled genome-wide shRNA screen that provides us with a list of HER2 synthetic lethal genes. By combining the analysis from RNAi loss-of-function screens with system biology interactome models, we have recently found that the activation of the JAK/STAT pathway is essential for HER2 mediated transformation, as we demostrate by knocking-down STAT3 expression either in vitro or in orthotopic mouse models. To investigate the mechanism mediating STAT3 activation, we compared the expression profile of parental and HER2 activated cells and found a strong upregulation of IL6 and its canonical receptor that was associated with an increase of IL6 in the media. Overall, our data shows that overexpression of HER2 in breast cancer cell lines leads to an increase in the expression and secretion of IL6, generating an autocrine loop that keeps JAK/STAT3 pathway constitutively active. To further study the transcriptional program regulated by STAT3 in HER2+ breast cancer cell lines, we analyzed the RNA profile in different conditions by using expression microarrays. Interestingly, our results included STAT3 as well as SOCS3, previously described targets of STAT3, but also two S100 family members: S100A8 and S100A9. Our data shows that the overexpression of these two genes is also essential for the transformation of HER2+ cells in vitro and in orthotopic mouse models.In order to translate our results to the clinical setting, we searched for compounds that could inhibit STAT3 activity in vivo. Ruxolitinib is a small-molecule inhibitor of JAK1 and JAK2 approved by the FDA for the treatment of patients with myelofibrosis and other myeloproliferative disorders, so we decieded to assess if Ruxolitnib was effective at inhibiting STAT3 signaling in our model. In fact, treatment with Ruxolitinib impairs in vitro transformation of cells that overexpress HER2 as shown by soft agar assays and 3D cultures in matrigel. More interestingly, oral administration of Ruxolitinib to mice significantly reduces the growth of the tumors implanted in the mammary fat pad.In summary, JAK/STAT pathway activation is essential for HER2-associated transformation suggesting this pathway inhibition as a promising therapeutic approach in HER2 positive tumors that do not respond to the conventional treatment.Note: This abstract was not presented at the meeting.Citation Format: Ruth Rodriguez Barrueco, Jiyang Yu, Mariano Alvarez, Veronica Castro, Netonia Marshall, Ruoxi Su, Laura Castro, Matthew Maurer, Andrea Califano, Jose M Silva. Characterizing the mechanism and the clinical relevance of the synthetic lethal interaction between STAT3 inhibition and HER2 overexpression in breast cancers. [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 2761. doi:10.1158/1538-7445.AM2014-2761

Recently, we demonstrated that the microRNA 424(322)/503 [miR-424(322)/503] cluster is transcriptionally controlled by transforming growth factor β (TGF-β) in the mammary epithelium. Induction of this microRNA cluster impacts mammary epithelium fate by regulating apoptosis and insulin-like growth factor 1 (IGF1) signaling. Here, we expanded our finding to demonstrate that miR-424(322)/503 is an integral component of the cell cycle arrest mediated by TGF-β. Mechanistically, we showed that after TGF-β exposure, increased levels of miR-424(322)/503 reduce the expression of the cell cycle regulator CDC25A. miR-424(322)/503-dependent posttranscriptional downregulation of CDC25A cooperates with previously described transcriptional repression of the CDC25A promoter and proteasome-mediated degradation to reduce the levels of CDC25A expression and to induce cell cycle arrest. We also provide evidence that the TGF-β/miR-424(322)/503 axis is part of the mechanism that regulates the proliferation of hormone receptor-positive (HR(+)) mammary epithelial cells in vivo.

Abstract HER2 is a receptor tyrosine kinase found overexpressed in 20-40% of breast tumors correlating with a malignant phenotype and worse prognosis. Different targeted therapies have been developed to specifically inhibit its activity such the monoclonal antibodies Trastuzumab and Pertuzumab or the small tyrosine kinase inhibitor Lapatinib. Unfortunately, a large number of these patient tumors eventually progress, acquiring resistance.. Our main goal is to identify which pathways have become essential for HER2 overexpressing cells but are not needed for normal cells to survive. To determine this, we performed a pooled genome-wide shRNA screen that provides us with a list of HER2 synthetic lethal genes. By combining the analysis from RNAi loss-of-function screens with system biology interactome models, we have recently found that the activation of the JAK/STAT pathway is essential for HER2 mediated transformation, as we demostrate by knocking-down STAT3 expression either in vitro or in orthotopic mouse models. To investigate the mechanism mediating STAT3 activation, we compared the expression profile of parental and HER2 activated cells and found a strong upregulation of IL6 and its canonical receptor that was associated with an increase of IL6 in the media. Overall, our data shows that overexpression of HER2 in breast cancer cell lines leads to an increase in the expression and secretion of IL6, generating an autocrine loop that keeps JAK/STAT3 pathway constitutively active. To further study the transcriptional program regulated by STAT3 in HER2+ breast cancer cell lines, we analyzed the RNA profile in different conditions by using expression microarrays. Interestingly, our results included STAT3 as well as SOCS3, previously described targets of STAT3, but also two S100 family members: S100A8 and S100A9. Our data shows that the overexpression of these two genes is also essential for the transformation of HER2+ cells in vitro and in orthotopic mouse models. In order to translate our results to the clinical setting, we searched for compounds that could inhibit STAT3 activity in vivo. Ruxolitinib is a small-molecule inhibitor of JAK1 and JAK2 approved by the FDA for the treatment of patients with myelofibrosis and other myeloproliferative disorders, so we decieded to assess if Ruxolitnib was effective at inhibiting STAT3 signaling in our model. In fact, treatment with Ruxolitinib impairs in vitro transformation of cells that overexpress HER2 as shown by soft agar assays and 3D cultures in matrigel. More interestingly, oral administration of Ruxolitinib to mice significantly reduces the growth of the tumors implanted in the mammary fat pad. In summary, JAK/STAT pathway activation is essential for HER2-associated transformation suggesting this pathway inhibition as a promising therapeutic approach in HER2 positive tumors that do not respond to the conventional treatment. Note: This abstract was not presented at the meeting. Citation Format: Ruth Rodriguez Barrueco, Jiyang Yu, Mariano Alvarez, Veronica Castro, Netonia Marshall, Ruoxi Su, Laura Castro, Matthew Maurer, Andrea Califano, Jose M Silva. Characterizing the mechanism and the clinical relevance of the synthetic lethal interaction between STAT3 inhibition and HER2 overexpression in breast cancers. [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 2761. doi:10.1158/1538-7445.AM2014-2761