Differentially expressed genes with AZD5153 treatment

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Protein modulation by AZD5153 in hematological cell lines

<p>Figure S1: Downregulation of CCR2, CD180, MYC and upregulation of HEXIM1 mRNAs by AZD5153 in cells can be detected at 4h; Figure S2: Other BRD4/BET compounds can also downregulate CCR2 and CD180 mRNAs in hematological tumor cell lines; Supplementary Table 1: Description of cell lines used in the RNAseq study; Supplementary Table 2: Information on qPCR assays used in these studies; Supplementary Table 3: More detailed information for each healthy volunteer and patient sample.</p>

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

Significant research in the field of reproductive endocrinology and infertility has been devoted to improving pregnancy rates in IVF. Many factors have been extensively examined all with the goal of improving pregnancy rates. However, even with a genetically normal embryo there is still a 40-70% success rate. This likely indicates that there is another factor that has not yet been delineated affecting the embryo's ability to implant. The normal reproductive tract microbiome at the time of IVF transfer has still yet to be fully elucidated. Prior work has been published looking at 16S rRNA(Ribosomal RNA) which has a lower resolution and requires the bacteria to be present in a database for it to be identified. Shotgun metagenomic sequencing looks at the all the bacteria present and can better differentiate between closely related sub-species. We sought to obtain shotgun sequencing of the reproductive tract to determine what bacteria are present at time of IVF transfer. Prospective study from 2018-2020 at a private fertility clinic. Patients undergoing frozen blast embryo transfer (FET) of an euploid embryo were screened for enrollment. Patients were excluded for uterine factor infertility. A swab of the vagina, cervix, and the tip of the transfer catheter were obtained and sent for shallow shotgun metagenomic sequencing. Primary outcome was most abundant taxa on cervix and vagina. Secondary outcome included Ongoing pregnancy/Live birth rate, clinical pregnancy rate and miscarriage rate. 12 patients were enrolled in the pilot study. 11 underwent an FET with a euploid blast. 8 transfers resulted in a clinical pregnancy, 7 transfers resulted in a Live Birth with 1 miscarriage. 3 transfers had failed implantation. Analysis of the shotgun sequencing in this small cohort was consistent with prior studies. Gardnerella (42%) was found in the one patient that miscarried, but also in one (38%) that had a successful pregnancy. Lactobacillus inners was seen in both successful and unsuccessful cycles. The top five most abundant genus found were Gardenerella, Lactobacillus, Leptospira, Paeniglutamicibacter, and streptococcus. Top five most abundant species found were Lactobacillus crispatus, L. jensenii, L. iners, L. amylovorus, Paeniglutamicibacter antarcticus and Gardnerella vaginalis. In the pilot results of an ongoing study, we were able to obtain shotgun sequencing data from the cervix and vagina at the time of IVF transfer. Lactobacillus as expected was the most commonly seen species. In this pilot study, the small number of participants limits our ability to draw any conclusions. However, we did establish the feasibility of obtaining specimens at time of IVF. We will continue this study with a larger number of patients to look for the relationship between IVF success rate and microbiome characteristics. We did not see any concerning changes in outcome from obtaining these samples at the time of IVF.

Purpose: There are several agents in early clinical trials targeting components of the adenosine pathway including A2AR and CD73. The identification of cancers with a significant adenosine drive is critical to understand the potential for these molecules. However, it is challenging to measure tumor adenosine levels at scale, thus novel, clinically tractable biomarkers are needed. Experimental Design: We generated a gene expression signature for the adenosine signaling using regulatory networks derived from the literature and validated this in patients. We applied the signature to large cohorts of disease from The Cancer Genome Atlas (TCGA) and cohorts of immune checkpoint inhibitor-treated patients. Results: The signature captures baseline adenosine levels in vivo (r(2) = 0.92, P = 0.018), is reduced after small-molecule inhibition of A2AR in mice (r(2) = -0.62, P = 0.001) and humans (reduction in 5 of 7 patients, 70%), and is abrogated after A2AR knockout. Analysis of TCGA confirms a negative association between adenosine and overall survival (OS, HR = 0.6, P < 2.2e(-16)) as well as progression-free survival (PFS, HR = 0.77, P = 0.0000006). Further, adenosine signaling is associated with reduced OS (HR = 0.47, P < 2.2e(-16)) and PFS (HR = 0.65, P = 0.0000002) in CD8(+) T-cell-infiltrated tumors. Mutation of TGF beta superfamily members is associated with enhanced adenosine signaling and worse OS (HR = 0.43, P < 2.2e(-16)). Finally, adenosine signaling is associated with reduced efficacy of anti-PD1 therapy in published cohorts (HR = 0.29, P = 0.00012). Conclusions: These data support the adenosine pathway as a mediator of a successful antitumor immune response, demonstrate the prognostic potential of the signature for immunotherapy, and inform patient selection strategies for adenosine pathway modulators currently in development.

Adenosine is a key suppressor of immune cell function. CD73 and other nucleotidases/phosphatases dephosphorylate and convert extracellular AMP into adenosine, which binds the A2A adenosine receptor (A2aR). There are several agents in early clinical trials targeting components of the adenosine pathway including A2AR and CD73. The identification of cancers with a significant adenosine drive is therefore critical to our understanding of the potential for these molecules. However, it is challenging to measure tumour adenosine levels on a pan-cancer scale. Here we describe the generation & validation of a gene expression signature that we use to characterise the pan-cancer landscape of adenosine signalling and its role within the tumour microenvironment. Natural Language Processing (NLP) was used to define a network of regulatory interactions between the A2aR receptor and downstream entities. 14 genes were identified as being positively regulated by A2aR and robustly expressed in tumours and so constitute the signature, which we demonstrate accurately recapitulates adenosine drive in mouse syngeneic models. We therefore applied the signature to a pan-cancer analysis of tumour transcriptomes from TCGA and found a significant negative impact on both overall survival (HR = 0.6, Cox PH p=0) and progression free survival (HR = 0.77, Cox PH p=0.0000006) in adenosine high tumours. An adenosine related survival deficit was also seen in CD8+ T-cell infiltrated tumours (OS HR = 0.47, Cox PH p=0; PFS HR = 0.65, Cox PH p=0.0000002). Studying adenosine signalling in the context of tumour genetics identified highly significant associations with TGFB super-family members including TGFBR2 (p=0.0000000013). These data support the adenosine pathway as a key mediator of a successful anti-tumour immune response, demonstrate the prognostic potential of the gene expression signature and could inform patient selection strategies for adenosine pathway modulators. Citation Format: Ben Sidders, Pei Zhang, Kelly Goodwin, Greg O'Connor, Alexandra Borodovsky, Jonathan Dry, Richard Woessner, Kris Sachsenmeier. The landscape of adenosine signaling in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 493.

CDK9 (cyclin-dependent kinase 9) regulates RNA transcription through its phosphorylation of RNA polymerase II and subsequent release from a “paused” elongation state, resulting in transcriptional activity. A transient inhibition of CDK9 activity will significantly impact transcripts with a short half-life, which includes the transcript for MCL1, an anti-apoptotic protein. AZD4573 is a highly potent and selective CDK9 inhibitor. We have previously reported that AZD4573 treatment results in dose-dependent inhibition of pSer2 of RNA polymerase II, reductions in MCL1 mRNA and protein, and an increase in cleaved caspase 3, resulting in cell death of hematological tumor cell lines and regressions in AML/DLBCL in vivo models [Cidado et. al., AACR Annual Meeting, Abstract 310 (2018)].

AbstractPurpose: AZD5153 is a novel BRD4/BET inhibitor with a distinctive bivalent bromodomain binding mode. To support its clinical development, we identified pharmacodynamic (PD) biomarkers for use in clinical trials to establish target engagement.Experimental Design: CCR2 and CD180 mRNAs, initially identified from whole transcriptome profiling, were further evaluated by quantitative PCR in hematologic cell lines, xenografts, and whole blood from rat, healthy volunteers, and patients with cancer. MYC and HEXIM1 mRNAs were also evaluated.Results: RNA-sequencing data showed consistent decreases in CCR2/CD180 expression across multiple hematologic cell lines upon AZD5153 treatment. Evaluation of dose dependence in MV4,11 cells confirmed activity at clinically relevant concentrations. In vivo downregulation of CCR2/CD180 mRNAs (>80%) was demonstrated in MV4,11 and KMS-11 xenograft tumors at efficacious AZD5153 doses. Consistent with in vitro rat blood data, an in vivo rat study confirmed greater inhibition of CCR2/CD180 mRNA in whole blood versus MYC at an efficacious dose. Finally, in vitro treatment of whole blood from healthy volunteers and patients with cancer demonstrated, in contrast to MYC, almost complete downregulation of CCR2/CD180 at predicted clinically achievable concentrations.Conclusions: Our data strongly support the use of CCR2 and CD180 mRNAs as whole blood PD biomarkers for BRD4 inhibitors, especially in situations where paired tumor biopsies are unavailable. In addition, they can be used as tumor-based PD biomarkers for hematologic tumors. MYC mRNA is useful as a hematologic tumor-based biomarker but suboptimal as a whole blood biomarker. Utility of HEXIM1 mRNA may be limited to higher concentrations. Clin Cancer Res; 23(4); 1025–35. ©2017 AACR.

Both experimental and clinical studies have shown that Spinal Cord Injury (SCI) has detrimental effects on remote organs and systems, yet there have been limited studies focusing on these more widespread effects. Despite attention to the pathophysiology of SCI with a goal of protection and repair, there is very little known about these gastrointestinal side‐effects or their effect on the gut microbiome. We sought to characterize long term effects of SCI on the gut by identifying changes in bacterial communication, microbiome composition, and inflammation. Bacterial quorum sensing molecules (QSMs) regulate various communication processes including growth, biofilm formation, and virulence factor production, thus providing information on the state of gut microbiota. Despite a repeatedly observed link between the intestinal microbiome and health, there is very limited data characterizing the gut microbiome in adult SCI patients with bowel dysfunction. We have employed a SCI rat model to investigate changes in the intestinal microbiome and gastrointestinal tract which may potentially contribute to or cause the GI dysfunction experienced by patients with SCI. Specifically, we determined the changes in the intestinal microbiota post‐SCI, most notably there was a significant increase in the relative abundance of Bifidobacteriaceae and Clostridiaceae post‐SCI, and investigated the potential underlying mechanisms via levels of pro‐inflammatory cytokines (IL1‐β, IL‐12, MIP‐2, and TNF‐α) and QSMs. This study continues to provide new information regarding the injury‐dependent changes on the gut microbiome and revealing microbiome‐host interactions that may contribute to the long term effects of SCI on the GI tract function. Identifying mechanisms behind these changes will aid in the development of new therapeutic strategies such as dietary modulation to improve quality of life for people living with SCI. Support or Funding Information University of Miami Miller School of Medicine, Miami Project to Cure Paralysis, Department of Biochemistry and Molecular Biology

Abstract 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.

Abstract Mcl-1 is an anti-apoptotic member of the Bcl-2 family of proteins and is frequently amplified or over-expressed in both solid tumors and hematological malignancies, suggesting that its activity may be important for the survival of cancer cells. CDK9 inhibition results in the down regulation of Mcl-1 mRNA and subsequent protein levels by inhibiting transcription and represents an indirect approach to targeting Mcl-1. Mcl-1 can also be targeted directly using an inhibitor that disrupts the Mcl-1 complexes to induce apoptosis. Using both molecular and pharmacological approaches, we sought to identify predictive biomarkers of Mcl1 dependency in sensitive NSCLC and multiple myeloma cell lines. Here we demonstrate that NSCLC cell lines lacking MCL1 gene copy number gains are not sensitive to siRNA mediated knockdown of Mcl-1 or Mcl-1 inhibition (cell line sensitivity to CDK9 or Mcl-1 inhibition is defined by potency and extent of caspase activation). However, the presence of a copy number alteration does not predict sensitivity to Mcl-1 inhibition. To better understand what the drivers of sensitivity are, we developed quantitative assays on the Peggy platform (a capillary based immunoassay platform by Protein Simple) to measure both Mcl-1 and Bcl-xL protein levels. Using these assays, we show a correlation between sensitivity to a CDK9 or Mcl1 inhibitor and Mcl-1 levels, as well as to the ratio of Mcl-1 to Bcl-xL protein in a NSCLC cell line panel. These findings were then extended into a panel of multiple myeloma cell lines. While somewhat broad activity for CDK9 or Mcl-1 inhibition is seen across the cell lines tested, a subset of the sensitive lines have MCL1 amplification and express high levels of Mcl-1 protein. Mcl-1 levels alone, however, do not predict for sensitivity across the panel and, similar to NSCLC, the ratio of Mcl1 to Bcl-xL expression has greater positive predictive value. These results provide the rationale for exploring Mcl-1 copy number alterations and Mcl-1 and Bcl-xL protein levels as predictive biomarkers for tumor response when treating with a CDK9 or Mcl-1 inhibitor in both NSCLC and multiple myeloma. Citation Format: Kristen McEachern, Greg O’Connor, Justin Cidado, Matthew Belmonte, Evan Barry, Hannah Dry, Paul Secrist, Lisa Drew. Predicting response to Mcl-1 targeting agents in NSCLC and multiple myeloma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3558.

Pim kinases have been shown to play a key role as downstream effectors of growth factor signaling in hematological malignancies. We have previously described AZD1208, a novel, orally bioavailable, highly selective pan Pim kinase inhibitor, undergoing clinical testing in AML. Specifically, we had demonstrated the anti-proliferative activity of AZD1208 in models of AML and had shown that AZD1208 treatment of AML cell lines results in the dose dependent reduction of pBAD at serine 112 and p4EBP1 at serine 65 as well as pp70S6K at threonine 389 and pS6 at serine 235/236, associated with global effects on protein translation (Keeton et al. Blood 2014).

Abstract Abstract 3720 Spleen tyrosine kinase (SYK) plays a key role in B cell receptor mediated survival in certain B cell malignancies including Diffuse Large B Cell Lymphoma (DLBCL). Therefore, targeting SYK represents an emerging therapeutic approach for the treatment of DLBCL. Indeed, fostamatinib, an orally available SYK inhibitor, has shown promising clinical activity in non-Hodgkin lymphoma (Friedberg et al. 2010 Blood 115: 2578). However, the overall response rate for novel targeted agents in unselected relapsed refractory DLBCL patients remains 25–30%, highlighting the opportunity for development of better treatment strategies. Here, we used preclinical models to study the mechanism underlining the efficacy of R406, the metabolic active form of fostamatinib. R406 was characterized in a heterogeneous panel of 17 DLBCL cell lines including both ABC and GCB subtypes. Overall, the cellular selectivity of R406, particularly in ABC-subtype DLBCL cell lines, was consistent with other BCR-targeted agents such as BTK inhibitor PCI-32765 (ibrutinib) and PI3 kinase δ inhibitor CAL-101. This strongly suggests that R406 functions through inhibition of BCR-mediated survival signaling. Furthermore, phospho-flow analysis and Western Blotting have demonstrated the effect of R406 on both basal and anti-IgG/M stimulated BCR signaling. Specifically, R406 decreases phosphorylation of proximal BCR pathway regulators BLNK, PLCγ2, as well as key players in downstream effect pathways such as ERK, AKT, RPS6, 4EBP and STAT3. To investigate whether the inhibition of BCR signaling by R406 affects cell viability, flow-cytometry based apoptosis analysis was employed. R406 induced apoptosis in sensitive cell lines in a dose-dependent manner. Mechanistically, treatment with R406 reduced MCL1 protein level and down-regulated Bfl-1 expression in sensitive, but not resistant, cell lines, which may partially contribute to the observed efficacy. Given the critical role of NFκB-dependent survival signaling in ABC subtype DLBCL, we then investigated the effect of R406 on this pathway by monitoring NF-κB target gene expression via quantitative real time PCR. Consistent with previous publication (Davis et al. 2010, Nature 463: 88), R406 significantly blocked chronic active BCR-induced NFκB signaling in sensitive cell lines with CD79A/B mutations, leading to downregulation of IL-6/IL-10 and subsequent suppression of JAK/STAT3 signaling. In contrast, we observed little effect of R406 in cell lines with downstream activating mutations of the NFκB pathway (such as CARD11, A20 mutations). Therefore, the molecular nature of NF-κB pathway lesions may serve as a predictive marker for R406 responsiveness in ABC subtype DLBCL cell lines. Furthermore, in exploration of a common indicator of sensitivity to R406 in both ABC and GCB subtypes, we have confirmed that response to R406 is dependent on surface expression of a functional BCR and presence of a intact BCR signaling cascade (Chen et al. 2008 Blood 111: 2230). These cells are thus characterized as “B-cell Receptor active” or BCR subtype. Work is currently underway to further elucidate the characteristics of the BCR subtype, which may serve as a general selective marker for R406's efficacy in DLBCL cell lines. In conclusion, we have demonstrated that R406 functions as a BCR antagonist and reduces viability in DLBCL through inhibition of NFκB-mediated survival signals and downregulation of MCL-1. In addition, we have confirmed in ABC subtype DLBCL cell lines, similar to other BCR-targeted agents, R406 sensitivity is correlated with CD79A/B mutations whereas A20 or CARD11 mutations render cells resistance. Eventually, we proposed that BCR classification may serve as a broader selection maker for all DLBCL cell lines. Disclosures: Zhang: AstraZeneca: Employment, Equity Ownership. Parmentier:AstraZeneca: Employment, Equity Ownership. Lai:AstraZeneca: Employment, Equity Ownership. O'Connor:AstraZeneca: Employment, Equity Ownership. Passino:AstraZeneca: Employment, Equity Ownership. Powell:AstraZeneca: Employment, Equity Ownership. Devereaux:AstraZeneca: Employment, Equity Ownership. Byth:AstraZeneca: Employment, Possible shareholder Other.