ALBERT

Description: Advances in Acute Myeloid Leukemia (AML) research have shown that the bone marrow microenvironment may distinctly vary across disease subtypes, and that this variation is associated with disease risk and response to conventional therapies. Novel therapies aimed at altering the tumor microenvironment, such as T-cell redirection, CAR-T and checkpoint inhibition, are emerging as promising treatment options for AML patients; however, there remains a critical need to determine how response to immune modulation may vary within different subsets of AML. Thus, in collaboration with the Beat AML Consortium, we carried out comprehensive mass cytometry profiling of patient bone marrow samples of nearly 100 Beat AML subjects and characterized their ex vivo response to several immune modulators. As a complement to this study, we leveraged the Beat AML Consortium dataset (including next-generation sequencing, functional cell-based assays, small molecule screening and clinical information) to investigate connections between disease subtype, immune function and clinical outcome. The mass cytometry time of flight (CyTOF) immune profiling, combined with matched genomic, cytogenetic, and outcome data from the same subjects, provided a unique opportunity to investigate features of the immune environment at single-cell resolution and test for their association with clinical covariates in a large treatment-naïve cohort. Interestingly, flow cytometry analysis of T-cells isolated from patient bone marrow showed a distinct subset of AML subjects with highly proliferative T-cells and a group of AML subjects with non-proliferative T-cells. To characterize molecular determinants of T-cell function in the AML microenvironment, we compared the transcriptional profiles of tumor specimens from subjects within these two groups. The data revealed a distinct set of differentially expressed genes associated with T-cell proliferation; pathway enrichment analysis indicated that these genes were involved in leukocyte migration, inflammation and response to hypoxia. Genes related to immune function were also enriched, likely due to the presence of immune cell infiltrates and stromal cells in addition to tumor cells from the AML specimens used for RNA-Seq. To estimate the extent of immune and stromal cells in the AML bone marrow, we next computed the approximate cellularity of the RNA-Seq samples using the xCell algorithm. The results of this analysis indicated enrichment of several types of immune cells in the RNA-Seq specimens from the proliferator group, including monocytes, neutrophils and activated dendritic cells. These observations were validated by preliminary results of the CyTOF immune cell profiling of the same subjects. Ongoing work is focused on the biological interpretation of CyTOF data collected for these subjects, including evaluating the association of functional marker expression on T-cell and myeloid cell populations with T-cell proliferation. Furthermore, we are exploring the functional impact of variation in T-cell fitness and immune cell composition on response to several immune modulators in a series of ex vivo experiments using Beat AML patient samples. Initial findings suggest that for a subset of patients, low baseline levels of T-cell proliferation did not prevent response to immune modulation. We are interrogating the Beat AML dataset for common molecular features of patients in this responder group. Overall, this study evaluates determinants of immune function and variation within the tumor microenvironment of AML patients to advance current knowledge of AML disease biology and to assess the impact of immune fitness on response to immune modulation. These results will contribute to early target identification and development, and importantly shed light on features of the AML bone marrow environment associated with response to therapy. Disclosures Brady: Janssen R&D: Employment. Soong:Janssen R&D: Employment. Lind:Celgene: Research Funding; Monojul: Research Funding; Amgen: Research Funding; Janssen Pharmaceutical R&D: Research Funding; Fluidigm: Honoraria. Schaffer:Janssen Research & Development: Employment, Equity Ownership. Hodkinson:Janssen R&D: Employment. Adams:Janssen Pharmaceutical R&D: Employment. Abraham:Janssen R&D: Employment. Safabakhsh:Janssen R&D: Employment. Tyner:AstraZeneca: Research Funding; Aptose: Research Funding; Array: Research Funding; Genentech: Research Funding; Constellation: Research Funding; Gilead: Research Funding; Incyte: Research Funding; Janssen: Research Funding; Takeda: Research Funding; Vivid Biosciences: Membership on an entity's Board of Directors or advisory committees. Druker:Aileron Therapeutics: Consultancy; MolecularMD: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Oregon Health & Science University: Patents & Royalties; Aptose Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Cepheid: Consultancy, Membership on an entity's Board of Directors or advisory committees; McGraw Hill: Patents & Royalties; Vivid Biosciences: Membership on an entity's Board of Directors or advisory committees; GRAIL: Consultancy, Membership on an entity's Board of Directors or advisory committees; Blueprint Medicines: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Bristol-Meyers Squibb: Research Funding; ARIAD: Research Funding; Novartis Pharmaceuticals: Research Funding; ALLCRON: Consultancy, Membership on an entity's Board of Directors or advisory committees; Third Coast Therapeutics: Membership on an entity's Board of Directors or advisory committees; Leukemia & Lymphoma Society: Membership on an entity's Board of Directors or advisory committees, Research Funding; Beta Cat: Membership on an entity's Board of Directors or advisory committees; Millipore: Patents & Royalties; Celgene: Consultancy; Gilead Sciences: Consultancy, Membership on an entity's Board of Directors or advisory committees; Patient True Talk: Consultancy; Amgen: Membership on an entity's Board of Directors or advisory committees; Fred Hutchinson Cancer Research Center: Research Funding; Monojul: Consultancy; Henry Stewart Talks: Patents & Royalties. Huang:Janssen R&D: Employment, Equity Ownership.

Description: Background: Immunoglobulin (IGH, IGL, IGK) and non-immunoglobulin (PVT1, TXNDC5, FAM46C, DUSP22, etc.) enhancers hijacking by variable genes (MYC, MAF, MAFB, CCND1/2/3, MMSET, IRF4) is a recognized oncogenic driver event in MM. However, the identity of the transcription factors (TFs) or transcriptional regulatory complexes binding and regulating the activity of these enhancers remains to be fully elucidated and may yield valuable therapeutic targets. As such the discovery of the BET family member BRD4 as the master histone acetyl mark reader at enhancers loci regulating MYC lead to promising therapeutic developments in MM and numerous other cancers. Immunomodulatory drugs (IMiDs) promote the proteasomal degradation of IKAROS (IKZF1) and AIOLOs (IKZF3) leading to the transcriptional repression of MYC and the suppression of MM cells survival and proliferation. However, acquired resistance to IMIDs and the loss of the transcriptional repression of MYC are nearly universal and occur in spite of sustained IKZF1/3 degradation suggesting that transcriptional rewiring may be sustaining hijacked enhancers activity and transcription of driver oncogenes. Methods and Results: In order to define how IMiDs repress MYC transcription, we first defined IKZF1, BRD4, the lysine acetyl transferase P300 and the mediator complex subunit MED1 mapping within the MM genome using ChIPseq. In MM cell lines (MM1S, RPMI8226, ARP1 and AMO1), IKZF1 predominantly mapped to intronic and intergenic loci which are typically enriched with enhancer and superenhancer elements. Indeed, IKZF1 mapping to the genome nearly completely (96.5%) overlapped that of P300, MED1 and BRD4 co-occupied enhancer and superenhancer loci. We also confirmed that in the MM1S sensitive cell lines IMiDs (lenalidomide 10 μM, 24h) exposure efficiently depleted IKZF1, BRD4, P300 and MED1 at enhancer loci with ensuing MYC (and MAF) downregulation. In contrast, in resistant cell lines (RPMI8226) and in spite efficient IKZF1 displacement, BRD4, P300 and MED1 were retained at the oncogenic enhancer (IGLL5) driving MYC (and MAF). These findings lead us to postulate that in IMiDs resistant cells retention of BRD4 and MED1 at oncogenic enhancers in the absence of IKZF1 likely results from rewiring of the TFs regulating MYC. To identify TFs that may co-localize with BRD4 and IKZF1, we analyzed the enrichment of DNA motifs at IKZF1and BRD4 co-occupied enhancers using the MEME suite motif-finding algorithms. This computational analysis revealed a strong enrichment at these MM enhancers of the GGAA motif recognized by the ETS family of TFs (P= 3.2 e-743) and other motifs boxes for the RUNX (P= 9.6 e-725), MYC/MYB ( P= 8.8 e-52) and interferon regulatory (IRF) (P= 3.1 e-293) TFs. We next confirmed that the ETS family TF ETV4 was indeed expressed in IMiDs resistant, but not sensitive, MM cell lines. ChiPseq occupancy profiles in IMiDs resistant RPMI8226 cell line revealed co-localization of ETV4 with IKZF1, P300 and BRD4. As predicted, lenalidomide treatment induced global depletion of IKZF1 but not ETV4 at BRD4 occupied enhancers in resistant cell lines (RPMI8226 and ARP1). Importantly, Cas9-mediated knock out of ETV4 in RPMI8226 cells sensitized them to lenalidomide with ensuing MYC downregulation and cell death. Confirming its role in MM, ETV4 transcript was indeed detectable in primary patients' samples in the CoMMpass data repository (ETV4 FPKM 〉1.0 in 112/724) and its expression was associated with significantly reduced survival outcomes (HR 0.64; P=0.0008). Similarly, high expression (top quartiles) of RUNX2 or MYB, TFs with enriched motifs at IKZF1 co-occupied enhancer loci, was also associated with decreased survival. Of note RNAseq analysis of paired patient samples pre- and post-IMiDs treatment (n=14 pairs) revealed significant upregulation of ETV4 at the time of acquired IMiDs resistance (7/14). Lastly transcriptome analysis of 101 patients enrolled in the RD arm (lenalidomide and dexamethasone) of the POLLUX trial (NCT02076009) confirmed the reduced survival of patients with top quartiles expression of ETV4 as well as MYB and RUNX2 (Fig.1) Conclusion: Transcriptional plasticity with expression of extra-lineage TFs such as the ETS family member ETV4 sustains oncogenic enhancers in MM overcoming IKAROS and AIOLOS dependency and promoting IMiDs resistance. Figure 1 Figure 1. Disclosures Neri: Celgene: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding. Soong: Jannsen: Employment. Chiu: Janssen: Employment. Bahlis: Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.