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Supplementary Figure 1. AZD5153 shows reduced BRD4 binding activity when BD2 function is abolished; Supplementary Figure 2. AZD5153 modulates MYC protein levels across hematologic cancer cell lines; Supplementary Figure 3. Gene Ontology (GO) analysis of the down-regulated transcripts by AZD5153 across all cell line types; Supplementary Figure 4. IC50 and percent maximal cell kill by AZD5153 and I-BET762 in five hematologic cell lines; Supplementary Figure 5. Tumor growth inhibition across six hematological xenograft Models

Protein modulation by AZD5153 in hematological cell lines

Cell line authentication information

The bromodomain and extraterminal (BET) protein BRD4 regulates gene expression via recruitment of transcriptional regulatory complexes to acetylated chromatin. Pharmacological targeting of BRD4 bromodomains by small molecule inhibitors has proven to be an effective means to disrupt aberrant transcriptional programs critical for tumor growth and/or survival. Herein, we report AZD5153, a potent, selective, and orally available BET/BRD4 bromodomain inhibitor possessing a bivalent binding mode. Unlike previously described monovalent inhibitors, AZD5153 ligates two bromodomains in BRD4 simultaneously. The enhanced avidity afforded through bivalent binding translates into increased cellular and antitumor activity in preclinical hematologic tumor models. In vivo administration of AZD5153 led to tumor stasis or regression in multiple xenograft models of acute myeloid leukemia, multiple myeloma, and diffuse large B-cell lymphoma. The relationship between AZD5153 exposure and efficacy suggests that prolonged BRD4 target coverage is a primary efficacy driver. AZD5153 treatment markedly affects transcriptional programs of MYC, E2F, and mTOR. Of note, mTOR pathway modulation is associated with cell line sensitivity to AZD5153. Transcriptional modulation of MYC and HEXIM1 was confirmed in AZD5153-treated human whole blood, thus supporting their use as clinical pharmacodynamic biomarkers. This study establishes AZD5153 as a highly potent, orally available BET/BRD4 inhibitor and provides a rationale for clinical development in hematologic malignancies. Mol Cancer Ther; 15(11); 2563–74. ©2016 AACR.

Differentially expressed genes with AZD5153 treatment

Syngeneic mouse tumor efficacy, STAT3, and gene expression changes

Patient STAT reductions and reproducibility of replicates

Patient sample details

Supplemental Methods. Methods used to generate supplemental data

Localized angiopoietin-2 (Ang2) expression has been shown to function as a key regulator of blood vessel remodeling and tumor angiogenesis, making it an attractive candidate for antiangiogenic therapy. A fully human monoclonal antibody (3.19.3) was developed, which may have significant pharmaceutical advantages over synthetic peptide-based approaches in terms of reduced immunogenicity and increased half-life to block Ang2 function. The 3.19.3 antibody potently binds Ang2 with an equilibrium dissociation constant of 86 pmol/L, leading to inhibition of Tie2 receptor phosphorylation in cell-based assays. In preclinical models, 3.19.3 treatment blocked blood vessel formation in Matrigel plug assays and in human tumor xenografts. In vivo studies with 3.19.3 consistently showed broad antitumor activity as a single agent across a panel of diverse subcutaneous and orthotopic xenograft models. Combination studies of 3.19.3 with cytotoxic drugs or anti–vascular endothelial growth factor agents showed significant improvements in antitumor activity over single-agent treatments alone with no apparent evidence of increased toxicity. Initial pharmacokinetic profiling studies in mice and nonhuman primates suggested that 3.19.3 has a predicted human half-life of 10 to 14 days. These studies provide preclinical data for 3.19.3 as a potential new antiangiogenic therapy as a single agent or in combination with chemotherapy or vascular endothelial growth factor inhibitors for the treatment of cancer. Mol Cancer Ther; 9(1); 145–56

Supplemental Figure 3. HGF and HGFAC expression in RCC-43b and RCC-47

Patient tumor gene expression data

DNA damage checkpoint kinases ATR and WEE1 are among key regulators of DNA damage response pathways protecting cells from replication stress, a hallmark of cancer that has potential to be exploited for therapeutic use. ATR and WEE1 inhibitors are in early clinical trials and success will require greater understanding of both their mechanism of action and biomarkers for patient selection. Here, we report selective antitumor activity of ATR and WEE1 inhibitors in a subset of non-germinal center B-cell (GCB) diffuse large B-cell lymphoma (DLBCL) cell lines, characterized by high MYC protein expression and CDKN2A/B deletion. Activity correlated with the induction of replication stress, indicated by increased origin firing and retardation of replication fork progression. However, ATR and WEE1 inhibitors caused different amounts of DNA damage and cell death in distinct phases of the cell cycle, underlying the increased potency observed with WEE1 inhibition. ATR inhibition caused DNA damage to manifest as 53BP1 nuclear bodies in daughter G1 cells leading to G1 arrest, whereas WEE1 inhibition caused DNA damage and arrest in S phase, leading to earlier onset apoptosis. In vivo xenograft DLBCL models confirmed differences in single-agent antitumor activity, but also showed potential for effective ATR inhibitor combinations. Importantly, insights into the different inhibitor mechanisms may guide differentiated clinical development strategies aimed at exploiting specific vulnerabilities of tumor cells while maximizing therapeutic index. Our data therefore highlight clinical development opportunities for both ATR and WEE1 inhibitors in non-GCB DLBCL subtypes that represent an area of unmet clinical need.Significance:ATR and WEE1 inhibitors demonstrate effective antitumor activity in preclinical models of DLBCL associated with replication stress, but new mechanistic insights and biomarkers of response support a differentiated clinical development strategy.

Supplemental Figure 2. MET copy number in RCC-43 tumors from passage 6 and 9, and in RCC-47 tumors from passage 5 and 10 by qPCR

Purpose: Papillary renal cell carcinoma (PRCC) is the second most common cancer of the kidney and carries a poor prognosis for patients with nonlocalized disease. The HGF receptor MET plays a central role in PRCC and aberrations, either through mutation, copy number gain, or trisomy of chromosome 7 occurring in the majority of cases. The development of effective therapies in PRCC has been hampered in part by a lack of available preclinical models. We determined the pharmacodynamic and antitumor response of the selective MET inhibitor AZD6094 in two PRCC patient-derived xenograft (PDX) models.Experimental Design: Two PRCC PDX models were identified and MET mutation status and copy number determined. Pharmacodynamic and antitumor activity of AZD6094 was tested using a dose response up to 25 mg/kg daily, representing clinically achievable exposures, and compared with the activity of the RCC standard-of-care sunitinib (in RCC43b) or the multikinase inhibitor crizotinib (in RCC47).Results: AZD6094 treatment resulted in tumor regressions, whereas sunitinib or crizotinib resulted in unsustained growth inhibition. Pharmacodynamic analysis of tumors revealed that AZD6094 could robustly suppress pMET and the duration of target inhibition was dose related. AZD6094 inhibited multiple signaling nodes, including MAPK, PI3K, and EGFR. Finally, at doses that induced tumor regression, AZD6094 resulted in a dose- and time-dependent induction of cleaved PARP, a marker of cell death.Conclusions: Data presented provide the first report testing therapeutics in preclinical in vivo models of PRCC and support the clinical development of AZD6094 in this indication. Clin Cancer Res; 21(12); 2811–9. ©2015 AACR.

Purpose:Danvatirsen is a therapeutic antisense oligonucleotide (ASO) that selectively targets STAT3 and has shown clinical activity in two phase I clinical studies. We interrogated the clinical mechanism of action using danvatirsen-treated patient samples and conducted back-translational studies to further elucidate its immunomodulatory mechanism of action.Experimental Design:Paired biopsies and blood samples from danvatirsen-treated patients were evaluated using immunohistochemistry and gene-expression analysis. To gain mechanistic insight, we used mass cytometry, flow cytometry, and immunofluorescence analysis of CT26 tumors treated with a mouse surrogate STAT3 ASO, and human immune cells were treated in vitro with danvatirsen.Results:Within the tumors of treated patients, danvatirsen uptake was observed mainly in cells of the tumor microenvironment (TME). Gene expression analysis comparing baseline and on-treatment tumor samples showed increased expression of proinflammatory genes. In mouse models, STAT3 ASO demonstrated partial tumor growth inhibition and enhanced the antitumor activity when combined with anti–PD-L1. Immune profiling revealed reduced STAT3 protein in immune and stromal cells, and decreased suppressive cytokines correlating with increased proinflammatory macrophages and cytokine production. These changes led to enhanced T-cell abundance and function in combination with anti–PD-L1.Conclusions:STAT3 ASO treatment reverses a suppressive TME and promotes proinflammatory gene expression changes in patients' tumors and mouse models. Preclinical data provide evidence that ASO-mediated inhibition of STAT3 in the immune compartment is sufficient to remodel the TME and enhance the activity of checkpoint blockade without direct STAT3 inhibition in tumor cells. Collectively, these data provide a rationale for testing this combination in the clinic.

Cytokine, T, and NK cell changes in CT26 tumors

Gating and antibodies for flow cytometry and CyTOF

Supplementary Table 1, Figure Legends 1-6 from A Human Monoclonal Anti-ANG2 Antibody Leads to Broad Antitumor Activity in Combination with VEGF Inhibitors and Chemotherapy Agents in Preclinical Models

Supplemental Fig S1- S6

Effects on cultured (human and mouse) immune cells

Methods Table: Antibodies

Gene expression signatures used in Figure 1

PARP proteins represent a class of post-translational modification enzymes with diverse cellular functions. Targeting PARPs has proven to be efficacious clinically, but exploration of the therapeutic potential of PARP inhibition has been limited to targeting poly(ADP-ribose) generating PARP, including PARP1/2/3 and tankyrases. The cancer-related functions of mono(ADP-ribose) generating PARP, including PARP6, remain largely uncharacterized. Here, we report a novel therapeutic strategy targeting PARP6 using the first reported PARP6 inhibitors. By screening a collection of PARP compounds for their ability to induce mitotic defects, we uncovered a robust correlation between PARP6 inhibition and induction of multipolar spindle (MPS) formation, which was phenocopied by PARP6 knockdown. Treatment with AZ0108, a PARP6 inhibitor with a favorable pharmacokinetic profile, potently induced the MPS phenotype, leading to apoptosis in a subset of breast cancer cells in vitro and antitumor effects in vivo. In addition, Chk1 was identified as a specific substrate of PARP6 and was further confirmed by enzymatic assays and by mass spectrometry. Furthermore, when modification of Chk1 was inhibited with AZ0108 in breast cancer cells, we observed marked upregulation of p-S345 Chk1 accompanied by defects in mitotic signaling. Together, these results establish proof-of-concept antitumor efficacy through PARP6 inhibition and highlight a novel function of PARP6 in maintaining centrosome integrity via direct ADP-ribosylation of Chk1 and modulation of its activity.Significance:These findings describe a new inhibitor of PARP6 and identify a novel function of PARP6 in regulating activation of Chk1 in breast cancer cells.

Supplemental Figure 1. In vitro activity of AZD6094, sunitinib, crizotinib, SGX-523, JNJ3887605, PHA-665752, and INC280 in AGS and GTL16 gastric cancer cells

This file contains all supplementary figures. Figure 1-7 provide additional analysis of genetic and cell features associated with AZD6738 and AZD1775 activity in DLBCL cell lines, including immunochemistry, immunoblots, immunofluorescence and flow cytometry data to further support the conclusions made from data presented in the main manuscript. Fig 8 provides tumour volume and body weight measurements for individual animals used in the in vivo studies.

Supplemental Table 1. Genes with copy number gain in RCC-43b and RCC-47

Supplemental materials

Supplemental Table 2. Mutations in RCC-43b and RCC-47

This file contains Supplementary Tables 1-6. Table S1 lists the growth inhibitory values of AZD6738 and cell doubling rates in DLBCL cell lines used in the study. Table S2 details the median replication fork velocities of DLBCL cell lines treated with DMSO and AZD6738 in Figure 3. Table S3 lists the statistical significance for DNA fibre analysis in Figure 3. Tables S4-S6 summarise the in vivo experiment groups used in the manuscript, including animal numbers in study, efficacy and body weight loss.

Purpose: Danvatirsen is a therapeutic antisense oligonucleotide (ASO) that selectively targets STAT3 and has shown clinical activity in two phase I clinical studies. We interrogated the clinical mechanism of action using danvatirsen-treated patient samples and conducted back-translational studies to further elucidate its immunomodulatory mechanism of action. Experimental Design: Paired biopsies and blood samples from danvatirsen-treated patients were evaluated using immunohistochemistry and gene-expression analysis. To gain mechanistic insight, we used mass cytometry, flow cytometry, and immunofluorescence analysis of CT26 tumors treated with a mouse surrogate STAT3 ASO, and human immune cells were treated in vitro with danvatirsen. Results: Within the tumors of treated patients, danvatirsen uptake was observed mainly in cells of the tumor microenvironment (TME). Gene expression analysis comparing baseline and on-treatment tumor samples showed increased expression of proinflammatory genes. In mouse models, STAT3 ASO demonstrated partial tumor growth inhibition and enhanced the antitumor activity when combined with anti–PD-L1. Immune profiling revealed reduced STAT3 protein in immune and stromal cells, and decreased suppressive cytokines correlating with increased proinflammatory macrophages and cytokine production. These changes led to enhanced T-cell abundance and function in combination with anti–PD-L1. Conclusions: STAT3 ASO treatment reverses a suppressive TME and promotes proinflammatory gene expression changes in patients' tumors and mouse models. Preclinical data provide evidence that ASO-mediated inhibition of STAT3 in the immune compartment is sufficient to remodel the TME and enhance the activity of checkpoint blockade without direct STAT3 inhibition in tumor cells. Collectively, these data provide a rationale for testing this combination in the clinic.

Mitogen-activated protein kinase (MAPK) pathway alterations comprise some of the most frequent mutations in newly diagnosed acute myeloid leukemia (AML). Moreover, MAPK pathway alterations are also emerging as potential mechanisms of resistance to targeted therapy in AML including FLT3 inhibitors and venetoclax. In an ex vivo pharmacologic analysis of primary AML samples, sensitivity to the MEK inhibitor selumetinib (ARRY-142886) was enriched in patient samples resistant to venetoclax. Clinical activity of MAPK pathway inhibitors such as selumetinib has been explored in AML but monotherapy responses were modest. Another MEK inhibitor, cobimetinib, is currently being tested in combination with venetoclax in AML. We sought to understand whether combining selumetinib with BH3 mimetics such as venetoclax would improve efficacy in AML models. We evaluated combination activity of selumetinib plus venetoclax or the MCL1 inhibitor AZD5991 in a panel of AML cell lines. Cells were exposed to a 6x6 matrix of both agents for 72hrs and then viability assessed using CellTiter-Glo. Combination benefit was assessed using highest single agent (HSA) analysis. Selumetinib+AZD5991 demonstrated strong combination benefit (HSA score >0.1, Emax >0.5) in 4/19 AML cell lines. Selumetinib+venetoclax showed strong combination activity in 6/19 lines. Three lines showed benefit with both combinations. Many of these cell lines harbor MAPK pathway mutations including OCI-AML5 (SOS1N233Y, NF1K1385R), ML-2 (KRASA146T), HL-60 (NRASQ61L), and Nomo-1 (KRASG13D). Selumetinib treatment also led to robust BIM induction in vitro. Nomo-1 xenografts were evaluated for in vivo sensitivity to venetoclax (100 mg/kg qd PO), 5-azacytidine (1mg/kg BID q8h 3days on/4days off IP), AZD5991 (30mpk bid q2h qw IV), selumetinib (10 mg/kg bid q8h PO), as well as combinations of venetoclax+5-aza, AZD5991+selumetinib, and venetoclax+selumetinib. Venetoclax and venetoclax+5-aza treatment were ineffective. Selumetinib monotherapy led to 63% tumor growth inhibition (TGI) but tumors eventually grew out through treatment. The combination of selumetinib+venetoclax slightly improved efficacy (81% TGI) but markedly delayed tumor outgrowth (selumetinib monotherapy arm reached mean tumor volume >1000 mm3 on day 17, selumetinib+venetoclax reached average of ~966 mm3 on day 28). Reducing venetoclax dose (30mg/kg qd) or frequency (100mg/kg 3days on/4days off) maintained most of the combination efficacy. Selumetinb+AZD5991 also strongly improved efficacy compared to either monotherapy (88% TGI, mean tumor volume did not exceed ~400mm3 by day 28). Together these data suggest potential for combining MEK inhibitors with BH3 mimetics in AML. Work is ongoing to further understand how scheduling impacts combination efficacy, evaluate the triple combination of selumetinib+BH3 mimetic+azacytidine, and assess efficacy in disseminated models. Citation Format: Courtney L. Andersen, Azadeh Cheraghchi-Bashi, Patricia Jaaks, Elizabeth A. Coker, Kathleen Burke, Justin Cidado, Paul Smith, Corinne Reimer, Raoul Tibes, Mathew Garnett, Jerome Mettetal. Combining selumetinib with BH3 mimetics enhances activity in MAPK-activated acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4024.

Dual Bcl-2/Bcl-xL inhibitors are expected to deliver therapeutic benefit in many haematological and solid malignancies, however, their use is limited by tolerability issues. AZD4320, a potent dual Bcl-2/Bcl-xL inhibitor, has shown good efficacy however had dose limiting cardiovascular toxicity in preclinical species, coupled with challenging physicochemical properties, which prevented its clinical development. Here, we describe the design and development of AZD0466, a drug-dendrimer conjugate, where AZD4320 is chemically conjugated to a PEGylated poly-lysine dendrimer. Mathematical modelling was employed to determine the optimal release rate of the drug from the dendrimer for maximal therapeutic index in terms of preclinical anti-tumour efficacy and cardiovascular tolerability. The optimised candidate is shown to be efficacious and better tolerated in preclinical models compared with AZD4320 alone. The AZD4320-dendrimer conjugate (AZD0466) identified, through mathematical modelling, has resulted in an improved therapeutic index and thus enabled progression of this promising dual Bcl-2/Bcl-xL inhibitor into clinical development., Claire Patterson et al. present the design and development of AZD0466, a drug-dendrimer conjugate, and use preclinical and mathematical models to determine the optimal release rate of the drug from the dendrimer carrier for maximal therapeutic index in terms of anti-tumour efficacy and cardiovascular tolerability. This study identifies this promising dual Bcl-2/Bcl-xL inhibitor for progression to clinical development.

Accumulation of extracellular adenosine within the microenvironment is a strategy exploited by tumors to escape detection by the immune system. Adenosine signaling through the adenosine 2A receptor (A2AR) on immune cells elicits a range of immunosuppressive effects which promote tumor growth and limit the efficacy of immune checkpoint inhibitors. Preclinical data with A2AR inhibitors have demonstrated tumor regressions in mouse models by rescuing T cell function; however, the mechanism and role on other immune cells has not been fully elucidated. Methods We report here the development of a small molecule A2AR inhibitor including characterization of binding and inhibition of A2AR function with varying amounts of a stable version of adenosine. Functional activity was tested in both mouse and human T cells and dendritic cells (DCs) in in vitro assays to understand the intrinsic role on each cell type. The role of adenosine and A2AR inhibition was tested in DC differentiation assays as well as co-culture assays to access the cross-priming function of DCs. Syngeneic models were used to assess tumor growth alone and in combination with alphaprogrammed death-ligand 1 (αPD-L1). Immunophenotyping by flow cytometry was performed to examine global immune cell changes upon A2AR inhibition. Results We provide the first report of AZD4635, a novel small molecule A2AR antagonist which inhibits downstream signaling and increases T cell function as well as a novel mechanism of enhancing antigen presentation by CD103+ DCs. The role of antigen presentation by DCs, particularly CD103+ DCs, is critical to drive antitumor immunity providing rational to combine a priming agent AZD4635 with check point blockade. We find adenosine impairs the maturation and antigen presentation function of CD103+ DCs. We show in multiple syngeneic mouse tumor models that treatment of AZD4635 alone and in combination with αPD-L1 led to decreased tumor volume correlating with enhanced CD103+ function and T cell response. We extend these studies into human DCs to show that adenosine promotes a tolerogenic phenotype that can be reversed with AZD4635 restoring antigen-specific T cell activation. Our results support the novel role of adenosine signaling as an intrinsic negative regulator of CD103+ DCs maturation and priming. We show that potent inhibition of A2AR with AZD4635 reduces tumor burden and enhances antitumor immunity. This unique mechanism of action in CD103+ DCs may contribute to clinical responses as AZD4635 is being evaluated in clinical trials with IMFINZI (durvalumab, αPD-L1) in patients with solid malignancies. Conclusion We provide evidence implicating suppression of adaptive and innate immunity by adenosine as a mechanism for immune evasion by tumors. Inhibition of adenosine signaling through selective small molecule inhibition of A2AR using AZD4635 restores T cell function via an internal mechanism as well as tumor antigen cross-presentation by CD103+ DCs resulting in antitumor immunity.

Mantle cell lymphoma (MCL) is an aggressive form of NHL where frequent relapse following standard therapies remains a serious concern, even for promising new treatments such as combinations of a BTK inhibitor with the selective Bcl2 inhibitor venetoclax. Previous studies have also shown that MCL cells develop resistance through tumor microenvironment interactions that increase levels of Mcl1, BclxL, and/or Bfl1. Given the ability of CDK9 inhibition to deplete Mcl1 and Bfl1 (Boiko et al 2021), we explored the potential of clinical-stage inhibitor AZD4573 to induce apoptosis in MCL cell lines and PDX models as a monotherapy or in combination with acalabrutinib. Currently, AZD4573 is being evaluated as a monotherapy in a first-in-human study for relapsed or refractory hematological malignancies (NCT03263637) as well as in combination with the BTK inhibitor, acalabrutinib, in patients with non-Hodgkin lymphoma (NHL) (NCT04630756). CDK9 is a serine/threonine kinase that mediates transcription elongation via phosphorylation of serine 2 of the RNA polymerase II carboxyl-terminal domain (pSer2-RNAP2). As previously shown, potent and selective inhibition of CDK9 by AZD4573 results in reduction of pSer2-RNAP2 levels leading to preferential depletion of labile proteins, including the Bcl2 family anti-apoptotic proteins Mcl1 and Bfl1 (as well as other known oncoproteins like Myc). This in turn drives rapid induction of apoptosis in a broad range of preclinical cancer models, particularly those derived from hematologic malignancies (Cidado et al 2020). Here, we used 7 MCL cell lines and 1 PDX organoid to assess the rapid apoptogenic potential of AZD4573 in vitro. Cleaved caspase-3 (CC3), a hallmark of apoptosis, was measured immediately following acute treatment (6h) using Caspase-Glo 3/7. Four models were sensitive to CDK9 inhibition (EC 50 < 100nM; max. CC3 > 50%) while 1 cell line exhibited intermediate sensitivity (EC 50 < 100nM; max. CC3 < 50%) and 3 others were resistant (EC 50 > 100nM; max. CC3 < 50%). Regardless of sensitivity, AZD4573 caused a dose- and time-dependent reduction of pSer2-RNAP2, Mcl1, and Myc, consistent with our prior reports. Most MCL cell lines are not responsive to BTK inhibition and, therefore, did not show combination benefit with AZD4573. We, therefore, chose to evaluate the in vivo activity of AZD4573 +/- acalabrutinib in 3 disseminated r/r MCL PDX models. In DFBL-44685, an acalabrutinib-unresponsive model harboring a CARD11 mutation, AZD4573 showed moderate activity, reducing MCL cells in all compartments analyzed (peripheral blood, bone marrow, and spleen) by >40% two weeks into treatment and increasing overall survival benefit (P80% and significantly increased survival over vehicle as well as monotherapy treatments (P50% and significantly increased survival over vehicle (P Our findings show that targeting CDK9 with AZD4573 can effectively induce apoptosis in a range of MCL cell lines and PDX models, including acalabrutinib-sensitive and -insensitive models as well as those expressing high levels of Bfl1. In 3 r/r MCL PDX models, single agent AZD4573 significantly reduced the tumor burden in the peripheral blood, bone marrow, and spleen of the affected mice, resulting in increased survival. Combination of AZD4573 with acalabrutinib resulted in greater anti-tumor activity than either monotherapy. Altogether, these data suggest that AZD4573, alone or in combination with acalabrutinib, could be an effective therapy for patients with r/r MCL. Figure 1 Figure 1. Disclosures Boiko: AstraZeneca: Current Employment, Current equity holder in publicly-traded company. Andersen: AstraZeneca: Current Employment, Current equity holder in publicly-traded company. Liang: AstraZeneca: Current Employment, Current equity holder in publicly-traded company. Dowdell: AstraZeneca: Current Employment, Current equity holder in publicly-traded company. Reimer: AstraZeneca: Current Employment, Current equity holder in publicly-traded company. Drew: AstraZeneca: Current Employment, Current equity holder in publicly-traded company. Townsley: AstraZeneca: Current Employment, Current equity holder in publicly-traded company. Cidado: AstraZeneca: Current Employment, Current equity holder in publicly-traded company.

We have established a novel assay to assess circulating tumor DNA (ctDNA) in mice engrafted with disseminated cell line and patient-derived xenografts (PDX) of hematologic malignancies. Disseminated models recapitulate many features of human disease, but engraftment in multiple tissues makes monitoring of disease and treatment response difficult. Existing assays also lack the sensitivity required to assess minimal residual disease (MRD). This novel ddPCR assay targets highly conserved human-specific regions of LINE-1 and HERV-K repeat elements resulting in exceptionally sensitive detection of shed human ctDNA. Initial data shows sensitivity of 0.8 haploid genome equivalents (one haploid genome is ~3.3pg of human DNA), whereas ctDNA monitoring assays currently on the market require 1,000 times more input DNA to reach a sensitivity of just 2-9 genome equivalents. Serial dilution experiments confirm this assay is suitable to detect increases in ctDNA over several orders of magnitude, while also establishing a minimum plasma input of 40uL. To validate the assay we assessed the ability of ctDNA to detect disease alongside traditional histology, bioluminescence imaging, and flow cytometry assays in 10 leukemia and lymphoma models. Levels of ctDNA correlated well with disease progression across models engrafting in bone marrow, spleen, liver, blood and other tissues. In all cases where early stage disease was analyzed, the ctDNA assay was able to detect disease earlier relative to other methods due to its increased sensitivity, and these data can be effectively used for randomization into treatment groups. We also assessed the utility of ctDNA for determining drug treatment response in mice engrafted with a disseminated mantle cell lymphoma PDX. Baseline ctDNA assessment was completed on day 11 post-engraftment confirming ctDNA levels above baseline, at which point mice were divided into 7 treatment groups: Vehicle, Acalabrutinib, and 5 combination arms of Acalabrutinib plus clinically used agents. After 4 weeks of treatment disease burden was assessed by flow cytometry of the bone marrow (femur) compared to ctDNA detected in plasma. Efficacy readouts from both assays agreed for all groups, while the ctDNA assay was able to identify a significant difference between the two best responding arms which appeared to have near complete responses when assessed by flow cytometry alone. The fold difference between the two best responding arms was 1.1 fold by flow cytometry and 3.6 fold by ctDNA, illuminating a greater disparity in MRD and relapse potential between these 2 groups. This novel ctDNA assay allows for robust experimental designs to answer questions about the depth and durability of treatment responses, as well as to identify early stage disease. Work is ongoing to explore extended monitoring for relapse and application to solid tumor models. Citation Format: Amanda L. Christie, Paul Labrousse, Courtney L. Andersen, Justine E. Roderick, Jacob Gordon, Deborah Lawson, Denise Hughes, Kimberly Maratea, Daniel Stetson, Brandon Willis, Andrew Bloecher, Corinne Reimer, Brian Dougherty. A novel circulating tumor DNA (ctDNA) assay enables monitoring of disease progression and treatment response in disseminated preclinical hematologic cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2957.

The complex interplay between the immune system and cancer cells is challenging to model in preclinical species. Many commonly mutated genes that influence tumorigenesis actively participate in the activation, recruitment or suppression of the immune system. A growing body of clinical findings indicates that defined oncogenic driver mutations correlate with immune contexture and associated immunotherapy responses. The goal of this study was to generate a hematopoietic stem cells (HSCs) transplanted humanized mice model using patient-derived xenografts with defined mutations to understand whether genetic alterations in the cancer cells can influence the tumor's immune landscape. Loss of function in the tumor suppressor gene STK11/LKB1 are observed in 5-30% of NSCLC. Patients with STK11 mutations do not typically respond to immune checkpoint blockade, and analysis of patient tumor biopsies indicates that these tumors are poorly infiltrated by immune cells such as T cells and dendritic cells, but with higher density of suppressive myeloid cells and associated cytokines. Therefore, there is an interest to understand ways to improve IO responses in these patients. To determine whether we could recapitulate this biology in a preclinical model, we implanted three STK11mut and one wild type PDX on humanized mice generated from six cord blood donors, and compared the immune infiltration in these tumors. Our results demonstrate efficient engraftment of human immune cells in the peripheral blood (53.1%), spleen (64.2%), and bone marrow (59.3%) of humanized mice (n=29, animals). Along with T and B cells, myeloid immune populations such as monocytes, macrophages and dendritic cells, which are absent in the previous generation of humanized mice, were present in the peripheral blood (monocytes 4.7%, dendritic cells 7.2% ) and bone marrow (macrophage 27.8%, neutrophils 21.6% and 10.2% dendritic cells ) of humanized mice. Tumor human immune subpopulation cells were significantly different (One-way Anova analysis) between the STK11mut (n=3) vs. wild type (n=1) humanized PDXs models (n=7 animals/PDX model). Three STK11mut tumors had low percentage of human CD45+ leukocytes infiltration (STK11mut; 0.9, 1.1 and 2.3% vs. wt; 6.6%) We also found a significantly reduced percentage of CD8+ cytotoxic T cells (STK11mut; 5.6, 3.2, 3.1% vs. wt; 9.6%) and dendritic cells subsets (STK11mut; 3.6, 2.2, 3.1% vs. wt; 5.6%) in the STK11mut PDXs implanted on humanized mice. We have shown efficient engraftment of a multilineage human immune system in immunodeficient mice and a selective infiltration of human immune cells subsets in PDXs representing a key genetic segment of NSCLC. Our novel humanized PDX model can recapitulate human tumor immune reconstitution, providing a valuable opportunity to evaluate the benefit of immunomodulatory therapies and personalize immune intervention strategies. Citation Format: Maneesh Singh, Laura Bradshaw, Laura B. Prickett, Griffin Matthew, Maryann San Martin, Noel Monks, Lisa Drew, Simon T. Barry, Corinne Reimer, Theresa Proia. The genetic makeup of patient-derived xenografts shapes the immune landscape of humanized mice tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1813.

Gastrointestinal stromal tumor (GIST) is the most common human sarcoma driven by mutations in KIT or platelet-derived growth factor α (PDGFRα). Although first-line treatment, imatinib, has revolutionized GIST treatment, drug resistance due to acquisition of secondary KIT/PDGFRα mutations develops in a majority of patients. Second- and third-line treatments, sunitinib and regorafenib, lack activity against a plethora of mutations in KIT/PDGFRα in GIST, with median time to disease progression of 4 to 6 months and inhibition of vascular endothelial growth factor receptor 2 (VEGFR2) causing high-grade hypertension. Patients with GIST have an unmet need for a well-tolerated drug that robustly inhibits a range of KIT/PDGFRα mutations. Here, we report the discovery and pharmacological characterization of AZD3229, a potent and selective small-molecule inhibitor of KIT and PDGFRα designed to inhibit a broad range of primary and imatinib-resistant secondary mutations seen in GIST. In engineered and GIST-derived cell lines, AZD3229 is 15 to 60 times more potent than imatinib in inhibiting KIT primary mutations and has low nanomolar activity against a wide spectrum of secondary mutations. AZD3229 causes durable inhibition of KIT signaling in patient-derived xenograft (PDX) models of GIST, leading to tumor regressions at doses that showed no changes in arterial blood pressure (BP) in rat telemetry studies. AZD3229 has a superior potency and selectivity profile to standard of care (SoC) agents-imatinib, sunitinib, and regorafenib, as well as investigational agents, avapritinib (BLU-285) and ripretinib (DCC-2618). AZD3229 has the potential to be a best-in-class inhibitor for clinically relevant KIT/PDGFRα mutations in GIST.

Accumulation of extracellular adenosine within the microenvironment is a strategy exploited by tumors to escape detection by the immune system. Adenosine signaling through the adenosine 2A receptor (A2AR) on immune cells elicits a range of immunosuppressive effects which promote tumor growth and limit the efficacy of immune checkpoint inhibitors. Preclinical data with A2AR inhibitors have demonstrated tumor regressions in mouse models by rescuing T cell function; however, the mechanism and role on other immune cells has not been fully elucidated.MethodsWe report here the development of a small molecule A2AR inhibitor including characterization of binding and inhibition of A2AR function with varying amounts of a stable version of adenosine. Functional activity was tested in both mouse and human T cells and dendritic cells (DCs) in in vitro assays to understand the intrinsic role on each cell type. The role of adenosine and A2AR inhibition was tested in DC differentiation assays as well as co-culture assays to access the cross-priming function of DCs. Syngeneic models were used to assess tumor growth alone and in combination with alphaprogrammed death-ligand 1 (αPD-L1). Immunophenotyping by flow cytometry was performed to examine global immune cell changes upon A2AR inhibition.ResultsWe provide the first report of AZD4635, a novel small molecule A2AR antagonist which inhibits downstream signaling and increases T cell function as well as a novel mechanism of enhancing antigen presentation by CD103+ DCs. The role of antigen presentation by DCs, particularly CD103+ DCs, is critical to drive antitumor immunity providing rational to combine a priming agent AZD4635 with check point blockade. We find adenosine impairs the maturation and antigen presentation function of CD103+ DCs. We show in multiple syngeneic mouse tumor models that treatment of AZD4635 alone and in combination with αPD-L1 led to decreased tumor volume correlating with enhanced CD103+ function and T cell response. We extend these studies into human DCs to show that adenosine promotes a tolerogenic phenotype that can be reversed with AZD4635 restoring antigen-specific T cell activation. Our results support the novel role of adenosine signaling as an intrinsic negative regulator of CD103+ DCs maturation and priming. We show that potent inhibition of A2AR with AZD4635 reduces tumor burden and enhances antitumor immunity. This unique mechanism of action in CD103+ DCs may contribute to clinical responses as AZD4635 is being evaluated in clinical trials with IMFINZI (durvalumab, αPD-L1) in patients with solid malignancies.ConclusionWe provide evidence implicating suppression of adaptive and innate immunity by adenosine as a mechanism for immune evasion by tumors. Inhibition of adenosine signaling through selective small molecule inhibition of A2AR using AZD4635 restores T cell function via an internal mechanism as well as tumor antigen cross-presentation by CD103+ DCs resulting in antitumor immunity.

PARP proteins represent a class of post-translational modification enzymes with diverse cellular functions. Targeting PARPs has proven to be efficacious clinically, but exploration of the therapeutic potential of PARP inhibition has been limited to targeting poly(ADP-ribose) generating PARP, including PARP1/2/3 and tankyrases. The cancer-related functions of mono(ADP-ribose) generating PARP, including PARP6, remain largely uncharacterized. Here, we report a novel therapeutic strategy targeting PARP6 using the first reported PARP6 inhibitors. By screening a collection of PARP compounds for their ability to induce mitotic defects, we uncovered a robust correlation between PARP6 inhibition and induction of multipolar spindle (MPS) formation, which was phenocopied by PARP6 knockdown. Treatment with AZ0108, a PARP6 inhibitor with a favorable pharmacokinetic profile, potently induced the MPS phenotype, leading to apoptosis in a subset of breast cancer cells in vitro and antitumor effects in vivo. In addition, Chk1 was identified as a specific substrate of PARP6 and was further confirmed by enzymatic assays and by mass spectrometry. Furthermore, when modification of Chk1 was inhibited with AZ0108 in breast cancer cells, we observed marked upregulation of p-S345 Chk1 accompanied by defects in mitotic signaling. Together, these results establish proof-of-concept antitumor efficacy through PARP6 inhibition and highlight a novel function of PARP6 in maintaining centrosome integrity via direct ADP-ribosylation of Chk1 and modulation of its activity. Significance: These findings describe a new inhibitor of PARP6 and identify a novel function of PARP6 in regulating activation of Chk1 in breast cancer cells.

Acute myeloid leukemia (AML) is an aggressive, heterogeneous malignancy. AML patients whose disease relapses on chemotherapy or are unfit for aggressive induction regimens have limited therapeutic options. Many patients benefit from the combination of venetoclax (BCL2i) and a hypomethylating agent (HMA) but this regimen is rarely curative. The addition of novel agents could provide improved benefit for relapsed/refractory patients. To identify such regimens, we screened a panel of 10 AML cell lines with combinations of venetoclax and novel targeted agents. The agents used spanned multiple mechanisms of action (e.g. DNA damage response, kinase signaling, pro-apoptotic agents) and are all in early clinical development. Cells were treated for 72hrs and viability was assessed by CellTiter-Glo. In several of the cell lines that were insensitive or partially sensitive to venetoclax (OCI-AML3, KG1a, MonoMac6, THP1), combinations with inhibitors of MCL1 (AZD5991), AURKB (AZD2811), and BRD4 (AZD5153) showed synergistic activity (Loewe synergy score >5, growth inhibition > 180%) (Table 1). We next asked if these combinations were active in patient-derived xenograft (PDX) models of AML. We established an ex vivo co-culture assay using the HS-5 bone marrow stromal cell line. AML PDX cells were isolated from mouse spleens and plated in 96-well format in direct co-culture with HS-5 cells or in HS-5-derived conditioned media. Cells were treated with three doses of each monotherapy and three doses of fixed ratio combination. Replicate screens using cells from individual mice on different days confirmed data were reproducible (r2=0.687) across animals engrafted with the same PDX. Drug response was similar between conditioned media and direct co-culture assays (r2=0.81). Venetoclax sensitivity varied across PDX models ex vivo. Notably, 2/5 PDX models screened (DFAM-68555 and DFAM-10360) were insensitive to both venetoclax and the combination of venetoclax + 5-azacytidine (HMA) ex vivo. Both models were established from untreated/1L patients and harbor TP53 mutations. Combination treatments did not add additional benefit over venetoclax monotherapy in the DFAM-10360 model. However, in DFAM-68555, AZD5153, AZD5991, and AZD2811 showed improved activity over venetoclax alone (67%, 54%, and 67% vs. 26% decrease in viability for venetoclax alone, respectively). Since combination strategies will likely be most impactful in patients refractory to or relapsed after venetoclax, we chose this venetoclax insensitive model to prioritize in vivo. To confirm the translatability of these findings, we designed a pilot in vivo study using DFAM-68555. Mice were randomized to receive vehicle, venetoclax + HMA, or venetoclax + AZD5153 when peripheral blood disease reached ~5% (hCD45+hCD33+ cells by flow cytometry). After two weeks of dosing, animals were sacrificed to evaluate disease burden in bone marrow (sternum), spleen, and peripheral blood. The model remained insensitive to venetoclax + HMA in vivo. The combination of AZD5153 with venetoclax decreased disease burden in blood and spleen compared to vehicle (30% and 42% hCD45+CD33+ cells by flow cytometry vs 70% and 95%, respectively) with similar efficacy seen by immunohistochemistry in the bone. Finally, we screened these venetoclax combinations in additional aggressive AML PDX models which were resistant or only partially responsive to venetoclax in vivo. Addition of AZD2811NP and AZD5991 to venetoclax was more effective than venetoclax alone and venetoclax + HMA in the bone marrow. The most active combination varied from model to model. Efficacy screening in additional models is ongoing to further build ex vivo to in vivo translation and prioritize development of specific combinations. Also ongoing is genomic and transcriptomic profiling of these PDXs to identify potential predictive biomarkers of combination activity. In summary, we developed an ex vivo screening platform to test clinically actionable combinations for activity in clinically relevant models. Using this platform and subsequent in vivo efficacy, we identified venetoclax combinations across multiple mechanisms (pro-apoptotic, cell cycle regulation, transcriptional regulation, DNA damage response) with activity in venetoclax-insensitive models. These results suggest potential therapeutic options to explore clinically for AML patients. Disclosures Andersen: AstraZeneca: Employment. Christie:AstraZeneca: Employment. Rosen:Astrazeneca: Employment. Maratea:AstraZeneca: Employment. Hattersley:AstraZeneca: Employment. Travers:AstraZeneca: Employment. Cidado:AstraZeneca: Employment. Pulukuri:AstraZeneca: Employment. Saeh:AstraZeneca: Employment. Clark:AstraZeneca: Employment, Equity Ownership. Reimer:AstraZeneca: Employment. Mettetal:AstraZeneca: Employment.

// Teeru Bihani 1 , Scott A. Ezell 1 , Brendon Ladd 1 , Shaun E. Grosskurth 1 , Anne Marie Mazzola 1 , Mark Pietras 1 , Corinne Reimer 1 , Michael Zinda 1 , Stephen Fawell 1 and Celina M. D’Cruz 1 1 AstraZeneca, R&D Boston, Waltham, MA Correspondence: Celina M. D’Cruz, email: // Keywords : everolimus, resistance, mTOR, MYC, BRD4 Received : October 29, 2014 Accepted : December 10, 2014 Published : December 11, 2014 Abstract Acquired resistance to PI3K/mTOR/Akt pathway inhibitors is often associated with compensatory feedback loops involving the activation of oncogenes. Here, we have generated everolimus resistance in ER+ breast cancer cells and in long-term estrogen deprived (LTED) models that mimic progression on anti-estrogens. This allowed us to uncover MYC as a driver of mTOR inhibitor resistance. We demonstrate that both everolimus resistance and acute treatment of everolimus can lead to the upregulation of MYC mRNA, protein expression and, consequently, the enrichment of MYC signatures as revealed by RNA sequencing data. Depletion of MYC resulted in resensitization to everolimus, confirming its functional importance in this setting. Furthermore, ChIP assays demonstrate that MYC upregulation in the everolimus resistant lines is mediated by increased association of the BRD4 transcription factor with the MYC gene. Finally, JQ1, a BRD4 inhibitor combined with everolimus exhibited increased tumor growth inhibition in 3D Matrigel models and an in vivo xenograft model. These data suggest that MYC plays an important role in mediating resistance to everolimus in ER+ and ER+/LTED models. Furthermore, given the regulation of MYC by BRD4 in this setting, these data have implications for increased therapeutic potential of combining epigenetic agents with mTOR inhibitors to effectively downregulate otherwise difficult to target transcription factors such as MYC.

Previous reports have demonstrated that select cancers depend on BRD4 to regulate oncogenic gene transcriptional programs. Here, we describe a novel role for BRD4 in DNA damage response (DDR). BRD4 associates with and regulates the function of pre-replication factor CDC6 and plays an indispensable part in DNA replication checkpoint signaling. Inhibition of BRD4 by JQ1 or AZD5153 resulted in a rapid, time-dependent reduction in CHK1 phosphorylation and aberrant DNA replication re-initiation. Furthermore, BRD4 inhibition sensitized cancer cells to various replication stress-inducing agents, and synergized with ATR inhibitor AZD6738 to induce cell killing across a number of cancer cell lines. The synergistic interaction between AZD5153 and AZD6738 is translatable to in vivo ovarian cell-line and patient-derived xenograft models. Taken together, our study uncovers a new biological function of BRD4 and provides mechanistic rationale for combining BET inhibitors with DDR-targeted agents for cancer therapy.

The bromodomain and extraterminal (BET) protein BRD4 regulates gene expression via recruitment of transcriptional regulatory complexes to acetylated chromatin. Pharmacological targeting of BRD4 bromodomains by small molecule inhibitors has proven to be an effective means to disrupt aberrant transcriptional programs critical for tumor growth and/or survival. Herein, we report AZD5153, a potent, selective, and orally available BET/BRD4 bromodomain inhibitor possessing a bivalent binding mode. Unlike previously described monovalent inhibitors, AZD5153 ligates two bromodomains in BRD4 simultaneously. The enhanced avidity afforded through bivalent binding translates into increased cellular and antitumor activity in preclinical hematologic tumor models. In vivo administration of AZD5153 led to tumor stasis or regression in multiple xenograft models of acute myeloid leukemia, multiple myeloma, and diffuse large B-cell lymphoma. The relationship between AZD5153 exposure and efficacy suggests that prolonged BRD4 target coverage is a primary efficacy driver. AZD5153 treatment markedly affects transcriptional programs of MYC, E2F, and mTOR. Of note, mTOR pathway modulation is associated with cell line sensitivity to AZD5153. Transcriptional modulation of MYC and HEXIM1 was confirmed in AZD5153-treated human whole blood, thus supporting their use as clinical pharmacodynamic biomarkers. This study establishes AZD5153 as a highly potent, orally available BET/BRD4 inhibitor and provides a rationale for clinical development in hematologic malignancies. Mol Cancer Ther; 15(11); 2563–74. ©2016 AACR.

// Ryan E. Henry 1,* , Evan R. Barry 1,* , Lillian Castriotta 1 , Brendon Ladd 1 , Aleksandra Markovets 1 , Garry Beran 2 , Yongxin Ren 3 , Feng Zhou 3 , Ammar Adam 1 , Michael Zinda 1 , Corinne Reimer 1 , Weiguo Qing 3 , Weiguo Su 3 , Edwin Clark 1 , Celina M. D’Cruz 1 and Alwin G. Schuller 1 1 AstraZeneca Pharmaceuticals PLC, Oncology Bioscience, Waltham, MA, USA 2 AstraZeneca Pharmaceuticals PLC, Oncology Bioscience, Alderley Park, UK 3 Hutchison Medi Pharma Ltd, Zhangjiang Hi-Tech Park, Shanghai, China * These authors have contributed equally to this work Correspondence to: Alwin G. Schuller, email: // Celina M. D’Cruz, email: // Keywords : MET, MYC, NSCLC, acquired resistance, savolitinib Received : January 14, 2016 Accepted : July 13, 2016 Published : July 26, 2016 Abstract Lung cancer is the most common cause of cancer death globally with a significant, unmet need for more efficacious treatments. The receptor tyrosine kinase MET has been implicated as an oncogene in numerous cancer subtypes, including non-small cell lung cancer (NSCLC). Here we explore the therapeutic potential of savolitinib (volitinib, AZD6094, HMPL-504), a potent and selective MET inhibitor, in NSCLC. In vitro , savolitinib inhibits MET phosphorylation with nanomolar potency, which correlates with blockade of PI3K/AKT and MAPK signaling as well as MYC down-regulation. In vivo , savolitinib causes inhibition of these pathways and significantly decreases growth of MET-dependent xenografts. To understand resistance mechanisms, we generated savolitinib resistance in MET -amplified NSCLC cell lines and analyzed individual clones. We found that upregulation of MYC and constitutive mTOR pathway activation is a conserved feature of resistant clones that can be overcome by knockdown of MYC or dual mTORC1/2 inhibition. Lastly, we demonstrate that mechanisms of resistance are heterogeneous, arising via a switch to EGFR dependence or by a requirement for PIM signaling. This work demonstrates the efficacy of savolitinib in NSCLC and characterizes acquired resistance, identifying both known and novel mechanisms that may inform combination strategies in the clinic.

Jak1/2 inhibition suppresses STAT3 phosphorylation that is characteristic of many cancers. Activated STAT3 promotes the transcription of factors that enhance tumor growth, survival, and angiogenesis. AZD1480 is a novel small molecule inhibitor of Jak1/2, which is a key mediator of STAT3 activation. This study examined the use of diffusion-weighted (DW) and dynamic contrast-enhanced (DCE) magnetic resonance imaging (MRI) biomarkers in assessing early tumor response to AZD1480. Cediranib (AZD2171), a vascular endothelial growth factor signaling inhibitor, was used as a comparator. Thirty mice were injected with Calu-6 lung cancer cells and randomized into the three treatment groups: AZD1480, cediranib, and sham. DW-MRI and DCE-MRI protocols were performed at baseline and at days 3 and 5 after treatment. The percent change from baseline measurements for K(trans), ADC, and v(e) were calculated and compared with hematoxylin and eosin (H&E), CD31, cParp, and Ki-67 histology data. Decreases in K(trans) of 29% (P < .05) and 53% (P < .05) were observed at days 3 and 5, respectively, for the cediranib group. No significant changes in K(trans) occurred for the AZD1480 group, but a significant increase in ADC was demonstrated at days 3 (63%, P < .05) and 5 (49%, P < .05). CD31 staining indicated diminished vasculature in the cediranib group, whereas significantly increased cParp staining for apoptotic activity and extracellular space by image analysis of H&E were present in the AZD1480 group. These imaging biomarker changes, and corresponding histopathology, support the use of ADC, but not K(trans), as a pharmacodynamic biomarker of response to AZD1480 at these time points.

Next-generation sequencing technologies have greatly expanded our understanding of cancer genetics. Antisense technology is an attractive platform with the potential to translate these advances into improved cancer therapeutics, because antisense oligonucleotide (ASO) inhibitors can be designed on the basis of gene sequence information alone. Recent human clinical data have demonstrated the potent activity of systemically administered ASOs targeted to genes expressed in the liver. We describe the preclinical activity and initial clinical evaluation of a class of ASOs containing constrained ethyl modifications for targeting the gene encoding the transcription factor STAT3, a notoriously difficult protein to inhibit therapeutically. Systemic delivery of the unformulated ASO, AZD9150, decreased STAT3 expression in a broad range of preclinical cancer models and showed antitumor activity in lymphoma and lung cancer models. AZD9150 preclinical activity translated into single-agent antitumor activity in patients with highly treatment-refractory lymphoma and non-small cell lung cancer in a phase 1 dose-escalation study.