Abstract: Infant acute lymphoblastic leukemia (ALL) is an orphan disease with unmet need for safe effective therapies. This is an urgent problem because conventional chemotherapies are ineffective and have life-threatening toxicities in infants. Although the MLL rearrangements occurring in 75% of cases are associated with poor outcome, survival is inferior whether MLL is rearranged or not. We recently reported that infant ALL proved sensitive to obatoclax mesylate (GeminX Pharmaceuticals; now an indirect, wholly owned subsidiary of Teva Pharmaceutical Industries Ltd.) in vitro regardless of poor prognostic features including MLL gene rearrangement. Moreover, we showed that the leukemia cell killing by obatoclax involved apoptosis, necroptosis and autophagy (Urtishak et al., Blood 2013). Therefore, the recent pharmaceutical abandonment of obatoclax led us to search for similarly acting drugs, the Results of which identified the well-known antipsychotic thioridazine as a candidate for potential repurposing. Methods Correlative analyses were performed between basal gene expression profiles at leukemia diagnosis and single agent obatoclax EC50 values from MTT assays in 47 cases of infant ALL from the Children's Oncology Group P9407 trial (25 MLL-AF4; 8 MLL-ENL; 7 other MLL-rearranged; 7 MLL-germline) in order to find a priori determinants of obatoclax sensitivity; significant genes were further studied by Ingenuity Pathway Analysis (IPA). A search for similarly acting compounds was conducted by Connectivity Map analysis of gene expression profiles of MLL-AF4 ALL cell lines after obatoclax treatment. MTT assays without and with cell death pathway inhibition, Western blot and flow cytometric cell death assays, and phosphoflow cytometric signaling analyses were utilized to investigate activity and target modulation by potential candidates. Results IPA identified significant correlations between basal gene expression of the mTOR and downstream intersecting eIF4/p70S6K signaling programs and obatoclax EC50 in all 47 primary cases of infant ALL, as well as in the subset of the 25 cases with MLL-AF4 rearrangements. Consistent with the relevance of this pathway in leukemia cell killing that was suggested by the basal gene expression profiles in the primary cases, the Connectivity Map analysis of obatoclax-treated cell lines for compound matching returned a number of highly ranked PI3K/AKT/mTOR signal transduction inhibitors as potential obatoclax substitutes. Three of the compounds (LY294002, wortmannin, thioridazine) were not only cytotoxic in MLL-AF4 ALL cell lines, but also they abrogated PI3K/AKT/mTOR signaling as indicated by robust inhibition of phosphorylated S6. Of these compounds, the phenothiazine derivative thioridazine, which has been used clinically for decades as a neuroleptic, was of high interest because of potential advantages of drug repurposing for more rapid drug advancement. Moreover, detailed flow cytometric and Western blot analyses, and MTT assays of thioridazine in the presence of cell death pathway inhibitors validated activation of all three cell death mechanisms in the MLL-AF4 ALL cell lines similarly to obatoclax. Conclusions Thioridazine is a well-known antipsychotic drug that also has recently recognized properties as a PI3K/AKT/mTOR signaling inhibitor and as an inhibitor of other pathways relevant to cancer. In MLL-AF4 ALL cell lines characterized by the most common chromosomal translocation in infant ALL, single-agent thioridazine is highly cytotoxic, robustly inhibits PI3K/AKT/mTOR signaling and, moreover, like obatoclax, demonstrates activity as a multi-cell-death pathway agonist. Further preclinical studies now are warranted to determine the extent to which thioridazine inhibits PI3K/AKT/mTOR signaling and causes leukemia cell killing in primary infant ALL cells in vitro and in vivo. The repurposing strategy that this drug may allow could have promise to streamline drug development in infant ALL where the need for new therapies is so urgent. Disclosures: No relevant conflicts of interest to declare.

Abstract: Background Acute myeloid leukemia (AML) is a molecularly and clinically heterogeneous hematological malignancy. Chemotherapy resistance is common, and relapse is the major cause of treatment failure. Although immunotherapy may be an attractive modality to exploit in patients with AML, the ability to predict the groups of patients and the types of cancer that will respond to immune targeting remains limited. Methods Immune gene expression profiling for the high-dimensional analysis of the immunological landscape of bone marrow (BM) samples from patients with newly-diagnosed (de novo) non-promyelocytic AML (n=387) was employed to analyze the tumor microenvironment (TME). We derived immune scores from mRNA expression levels and devised an RNA-based, quantitative metric of immune infiltration, as previously published (Danaher P, et al. JITC 2017 and 2018). The PanCancer IO360™ gene expression assay was used to profile BM samples collected prior to and during flotetuzumab (FLZ) treatment from 30 AML patients treated at the RP2D (500 ng/kg/day) in the CP-MGD006-01 clinical trial (NCT#02152956), primary refractory, n=23 or relapsed, n=7. IO360 score comparisons are presented as mean ± SD and significance was assessed by the Mann Whitney U test. Results Analysis of pre-treatment BM samples from de novo AML revealed distinct immune signature modules, reflecting the co-expression of genes associated with 1) an IFNγ-dominant TME, 2) adaptive immune responses, and 3) myeloid cell abundance. When considered in aggregate, the relative intensity of gene expression in the immune modules stratified BM samples into two subgroups, which will be herein termed immune-infiltrated and immune-depleted. When AML patients were dichotomized based on median immune scores, high versus low, a higher percentage of primary refractory patients was observed within the IFNγ high module (65.4% versus 34.6%; p=0.0022). In multivariate logistic regression, an IFNγ high profile (derived from the IFNγ-dominant module) was predictive of therapeutic resistance to induction chemotherapy, even more than ELN risk categories (AUROC = 0.815 versus 0.702 with ELN risk only). In a validation cohort, Beat AML series, the IFNγ high profile when compared to other clinically established prognostic indicators in AML, i.e. disease type (primary versus secondary), WBC count, patient age at diagnosis, and ELN risk categories was significantly more predictive of therapeutic resistance to induction chemotherapy (AUROC=0.921 versus 0.709 with ELN cytogenetic risk alone; two-tailed p value=0.002673). Similarly, a higher percentage of patients with an IFNγ high profile AML in the HOVON series failed to achieve CR in response to induction chemotherapy when compared to AML cases with an IFNγ low profile (27.2% versus 15.2%; p=0.0004). We hypothesized that higher expression of IFNγ inducible genes, while underpinning chemotherapy resistance, might identify AML patients who derive benefit from immunotherapy with FLZ. BM samples from 92% of patients with evidence of FLZ anti-leukemic activity (ALA), which was defined as either CR, CRh, CRi, PR or overall benefit ( & gt;30% reduction in BM blasts), had an immune infiltrated TME relative to non-responders (SD or PD). Interestingly, the IFNγ-signaling score was significantly higher in patients with chemotherapy-refractory AML compared with relapsed AML at time of FLZ treatment, and in individuals with evidence of ALA compared to non-responders (p & lt;0.0001). Additionally, another IFNγ-related score, the tumor inflammation signature (TIS), had strong predictive power of anti-leukemic activity to FLZ, with an AUROC value of 0.847 (p=0.001). FLZ also modified the TME, and on-treatment BM samples (available in 19 patients at the end of cycle 1) displayed increased expression of antigen presentation and immune activation genes relative to baseline, and had higher TIS scores (6.47±0.22 versus 5.93±0.15, p=0.0006), antigen processing machinery scores (5.67±0.16 versus 5.31±0.12, p=0.002), IFNγ signaling scores (3.58±0.27 versus 2.81±0.24, p=0.0004) and PD-L1 expression (3.43±0.28 versus 2.73±0.21, p=0.0062). Conclusions Our findings to date suggest that microenvironmental immune gene profiles could be used to inform the delivery of personalized immunotherapies to patients with IFNγ-dominant AML subtypes, and identify patients less likely to respond to cytotoxic chemotherapy. Disclosures Minden: Trillium Therapetuics: Other: licensing agreement. Hood:NanoString Technologies, Inc.: Employment. Church:NanoString Technologies, Inc.: Employment, Equity Ownership. Sullivan:NanoString Technologies, Inc.: Employment. Viboch:NanoString Technologies, Inc.: Employment. Warren:NanoString Technologies, Inc.: Employment. Liang:NanoString Technologies, Inc.: Employment. Cesano:NanoString Technologies, Inc.: Employment. Löwenberg:Chairman, Leukemia Cooperative Trial Group HOVON (Netherlands: Membership on an entity's Board of Directors or advisory committees; Agios Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Astellas: Membership on an entity's Board of Directors or advisory committees; Astex: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees; Up-to-Date", section editor leukemia: Membership on an entity's Board of Directors or advisory committees; CELYAD: Membership on an entity's Board of Directors or advisory committees; Chairman Scientific Committee and Member Executive Committee, European School of Hematology (ESH, Paris, France): Membership on an entity's Board of Directors or advisory committees; Elected member, Royal Academy of Sciences and Arts, The Netherlands: Membership on an entity's Board of Directors or advisory committees; Editorial Board "European Oncology & Haematology": Membership on an entity's Board of Directors or advisory committees; Clear Creek Bio Ltd: Consultancy, Honoraria; Hoffman-La Roche Ltd: Membership on an entity's Board of Directors or advisory committees; Frame Pharmaceuticals: Equity Ownership; Royal Academy of Sciences and Arts, The Netherlands: Membership on an entity's Board of Directors or advisory committees; Supervisory Board, National Comprehensive Cancer Center (IKNL), Netherland: Membership on an entity's Board of Directors or advisory committees. Tasian:Incyte Corportation: Research Funding; Gilead Sciences: Research Funding; Aleta Biotherapeutics: Membership on an entity's Board of Directors or advisory committees. Rettig:WashU: Patents & Royalties: Patent Application 16/401,950. Davidson-Moncada:MacroGenics, Inc.: Employment, Equity Ownership. DiPersio:Celgene: Consultancy; NeoImmune Tech: Research Funding; Macrogenics: Research Funding, Speakers Bureau; Incyte: Consultancy, Research Funding; Karyopharm Therapeutics: Consultancy; RiverVest Venture Partners Arch Oncology: Consultancy, Membership on an entity's Board of Directors or advisory committees; Cellworks Group, Inc.: Membership on an entity's Board of Directors or advisory committees; Amphivena Therapeutics: Consultancy, Research Funding; Magenta Therapeutics: Equity Ownership; WUGEN: Equity Ownership, Patents & Royalties, Research Funding; Bioline Rx: Research Funding, Speakers Bureau. Rutella:NanoString Technologies, Inc.: Research Funding; MacroGenics, Inc.: Research Funding.

Abstract: Acute myeloid leukemia (AML) is a molecularly and clinically heterogeneous hematological malignancy. Although immunotherapy may be an attractive modality to exploit in patients with AML, the ability to predict the groups of patients and the types of cancer that will respond to immune targeting remains limited. This study dissected the complexity of the immune architecture of AML at high resolution and assessed its influence on therapeutic response. Using 442 primary bone marrow samples from three independent cohorts of children and adults with AML, we defined immune-infiltrated and immune-depleted disease classes and revealed critical differences in immune gene expression across age groups and molecular disease subtypes. Interferon (IFN)–γ–related mRNA profiles were predictive for both chemotherapy resistance and response of primary refractory/relapsed AML to flotetuzumab immunotherapy. Our compendium of microenvironmental gene and protein profiles provides insights into the immuno-biology of AML and could inform the delivery of personalized immunotherapies to IFN-γ–dominant AML subtypes.

Abstract: Background Chemotherapy refractoriness and disease relapse continue to be significant obstacles to therapeutic success in AML. We have recently identified bone marrow (BM) transcriptomic profiles that stratify patients with AML into an immune-infiltrated and an immune-depleted subtype and that refine the accuracy of survival prediction in response to conventional chemotherapy beyond that afforded by cytogenetic and molecular prognosticators (Vadakekolathu J, et al. Sci. Transl. Med. 2020; 12: eaaz0463). Importantly, AML CD8+ T cells exhibit features of immune exhaustion and senescence (IES), including the upregulation of natural killer (NK) cell-associated transcripts, which persist only in chemotherapy non-responders (Knaus HA, et al. JCI Insight 2018; 3: e120974). The aim of the current study was to determine whether IES correlate with immune infiltration and with clinical outcomes in AML. Methods We used prior knowledge and gene set enrichment analysis (GSEA) to derive a 68-gene IES signature score (computed as the cohort-wide average of gene expression) from publicly available RNA-sequencing datasets (TCGA and Beat-AML Master Trial; 162 and 281 AML patients, respectively). The wet-lab AML cohorts used in this study included a total of 382 BM samples from children and adults with AML treated with curative intent. BMs were collected at time of diagnosis, complete remission (CR) and relapse (PMCC, SAL and CHOP series). BM RNAs were profiled on the nCounter platform using the PanCancer Immune Profiling Panel (NanoString Technologies, Seattle, WA). Results In the AML discovery cohort, the IES score significantly correlated with BM lymphoid infiltration (R=0.958) and with CD8+ T cell (R=0.88), cytotoxic T cell (R=0.90) and tumor inflammation signature scores (R=0.82, P & lt;0.0001 for all; Fig. 1A). The novel IES gene signature showed minimal overlap with previously discovered interferon (IFN)-related gene sets with prognostic relevance in AML (Fig. 1B) and was higher in TP53-mutated (P & lt;0.0001) and RUNX1-mutated (P=0.0071) compared with WT AML. Overall survival (OS) was significantly shorter in patients with higher than median IES scores (Fig. 1C). IES signature scores were also associated with worse clinical outcomes in Beat-AML cases (median OS of 10.6 and 21.1 months, respectively, in patients with higher and lower than median IES scores; Fig. 1D) and were higher in primary induction failures (PIF) compared with chemotherapy responders (Fig. 1E). The top IES genes associated with survival prediction both in TCGA and Beat-AML, although not overlapping with published IFN-related gene sets, were still significantly enriched in type I/II IFN signaling pathways and in molecular functions indicative of heightened ion-gated channel activity, as shown by protein-protein interaction network analyses. The above findings were validated in independent wet-lab cohorts comprising adults (PMCC series; n=290; SAL series; n=46) and children (CHOP series; n=46) with AML. In the ELN adverse risk subgroup (PMCC cohort), both relapse-free survival (RFS) and OS were significantly shorter in patients with higher than median compared with lower than median IES scores (median RFS time of 6.93 versus not reached [log-rank P=0.0053], and median OS of 10.5 months versus 18 months [log-rank P=0.0011] , respectively). In contrast, the IES signature score failed to stratify survival in patients with ELN favorable and intermediate risk. Finally, a pairwise comparison of matched diagnostic, CR and relapse BM samples (SAL series; n=22 patients and CHOP series; n=46 patients) showed significantly higher IES signature scores at time of CR, congruent with chemotherapy-induced acceleration of IES, and at time of post-chemotherapy relapse compared with disease onset (Fig. 1F). Notably, CD8A, TBX21, a Th1 transcription factor, and markers of NK cells and cytotoxic T lymphocytes, including KLRK1, KLRD1, KLRC2, GNLY, and granzymes, were among the top ranked immune genes associated with AML relapse (Fig. 1G). Conclusions Patients with immune-infiltrated AML exhibit features of IES, which may correlate with adverse-risk molecular features, chemotherapy refractoriness and shorter survival. Molecular circuits reflective of IES might also underpin AML relapse after conventional induction chemotherapy. IES T cells could be functionally rejuvenated by novel immunotherapies being investigated in AML. Figure 1 Disclosures Church: NanoString Technologies, Inc.: Current Employment. Davidson-Moncada:Macrogenics: Current Employment. Tasian:Incyte Corporation: Research Funding; Aleta Biotherapeutics: Membership on an entity's Board of Directors or advisory committees; Gilead Sciences: Research Funding. Gojo:Amphivena: Research Funding; Merck: Research Funding; Genentech: Research Funding; Amgen: Research Funding; BMS: Membership on an entity's Board of Directors or advisory committees. Luznik:WindMil Therapeutics: Patents & Royalties: Patent holder; AbbVie: Consultancy; Merck: Research Funding, Speakers Bureau; Genentech: Research Funding. Rutella:Kura Oncology: Research Funding; MacroGenics, Inc.: Research Funding; NanoString Technologies, Inc.: Research Funding.

Abstract: Not available.

Abstract: Acute myeloid leukemia (AML) is a genetically heterogeneous hematological malignancy with variable responses to chemotherapy. Although recurring cytogenetic abnormalities and gene mutations are important predictors of outcome, 50% to 70% of AMLs harbor normal or risk-indeterminate karyotypes. Therefore, identifying more effective biomarkers predictive of treatment success and failure is essential for informing tailored therapeutic decisions. We applied an artificial neural network (ANN)–based machine learning approach to a publicly available data set for a discovery cohort of 593 adults with nonpromyelocytic AML. ANN analysis identified a parsimonious 3-gene expression signature comprising CALCRL, CD109, and LSP1, which was predictive of event-free survival (EFS) and overall survival (OS). We computed a prognostic index (PI) using normalized gene-expression levels and β-values from subsequently created Cox proportional hazards models, coupled with clinically established prognosticators. Our 3-gene PI separated the adult patients in each European LeukemiaNet cytogenetic risk category into subgroups with different survival probabilities and identified patients with very high–risk features, such as those with a high PI and either FLT3 internal tandem duplication or nonmutated nucleophosmin 1. The PI remained significantly associated with poor EFS and OS after adjusting for established prognosticators, and its ability to stratify survival was validated in 3 independent adult cohorts (n = 905 subjects) and 1 cohort of childhood AML (n = 145 subjects). Further in silico analyses established that AML was the only tumor type among 39 distinct malignancies for which the concomitant upregulation of CALCRL, CD109, and LSP1 predicted survival. Therefore, our ANN-derived 3-gene signature refines the accuracy of patient stratification and the potential to significantly improve outcome prediction.

Abstract: 50 Background: Tumor phenotypes are dictated not only by the neoplastic cell component, but also by the tumor microenvironment (TME), which includes immune and inflammatory cells. We characterized the immune ecosystem of the bone marrow (BM) TME in patients with acute myeloid leukemia (AML) to correlate transcriptomic and proteomic profiles with patient outcome. Methods: We used the nCounter system (NanoString Technologies) to characterize BM specimens from 42 children and 28 adults with AML. Ninety BM samples (63 from de novo AML, 18 from AML in complete remission [CR] and 9 from relapsed AML) were analyzed using the RNA Pan-Cancer Immune Profiling Panel. In situ leukemia-immune system interactions were visualized using Digital Spatial Profiling (DSP). Results: Hierarchical clustering identified subgroups with heightened expression of lymphocyte-associated genes, including NK cell and cytotoxicity genes (“immune-enriched” samples). They also expressed CD8A, IFNG, FOXP3, the cell chemoattractants CXCL9, CXCL10, and inhibitory molecules including IDO1 and the immune checkpoints LAG3, CTLA4 and PD-L1. Conversely, ‘immune-depleted’ samples over-expressed genes associated with mast cell functions and CD8 T-cell exhaustion and showed low expression of T-cell and B-cell genes. Importantly, relapse-free survival (RFS) and overall survival (OS) were significantly shorter in patients with “immune-enriched” compared with “immune-depleted” AMLs (2.2 vs. 18.3 months, p = 0.0064, and 6.3 vs. 22.4 months, p = 0.017, respectively). DSP of the BM TME showed the co-localization of CD8 T cells with FoxP3 Treg cells and PD-L1- and VISTA-expressing cell types, an immune landscape which is consistent with the establishment of adaptive immune resistance mechanisms of immune escape. Conclusions: Our study has identified heterogeneous immune profiles in children and adults with AML. From a clinical standpoint, ‘immune enriched’ AMLs might be amenable to immunotherapy approaches tailored to the BM microenvironment, including blockade of co-inhibitory molecules and/or small-molecule IDO1 inhibitors. Grant support: Roger Counter Foundation, UK; Qatar National Research Fund (#NPRP8-2297-3-494).