Abstract: Introduction N 6-methyladenosine (m6A) is the most abundant internal modification in messenger RNA (mRNA) mainly occurring at consensus motif of G[G 〉 A]m6AC[U 〉 A 〉 C]. Despite the functional importance of m6A modification in various fundamental bioprocesses, the studies of m6A modification in cancer, especially in leukemia have largely been limited.Fat mass and obesity-associated protein (FTO), the first RNA demethylase,was known to be robustly associated with increased body mass and obesity in humans. However, the impact of FTO, especially as a RNA demethylase, in cancer development and progression has yet to be investigated. Acute myeloid leukemia (AML) is one of the most common and fatal forms of hematopoietic malignancies with distinct geneticabnormalities and variable response to treatment.Here, we aim to definethe roleof FTO as an m6A demethylase in AML. Methods To access the potential effect of FTO, we analyzed its expression in AML patients with distinct genetic mutations. To determine the influence of FTO on transformation capacity/ cell viability and leukemogenesis, colony-forming/replating assay (CFA), MTT assays, cell apoptosis and bone marrow transplantation (BMT) were carried out. To identify potential targets of FTO, transcriptome-wide m6A-seq and RNA-seq were performed. To evaluate the function of FTO on m6A modification and mRNA metabolism,m6A dot blot, gene-specific m6A qPCR assays and RNA stability assays were conducted. To elucidate whether FTO-mediated regulation of its targets depends on its demethylase activity, gene-specific m6A qPCR assays and luciferase reporter and mutagenesis assays were carried out. To investigate the potential roles of FTO and its targets in hematopoiesis, ATRA-induced APL cell differentiation was used. Results In analysis of AML datasets, we found FTO is highly expressed in AMLs with t(11q23)/MLL-rearrangements, t(15;17)/PML-RARA, FLT3-ITD and/or NPM1 mutations. Lentivirus-induced expression of wild-type FTO, but not mutant FTO (carrying two point mutations, H231A and D233A , which disrupt its enzymatic activity), significantly enhanced colony forming activities, promoted cell proliferation/transformation, restricted cell apoptosis and decreased global mRNA m6A levelin vitro. Forced expression of Fto significantly (p 〈 0.05; log-rank test) accelerated MLL-AF9-induced leukemogenesis and decreased global m6A level in leukemic BM cells. The opposite is true when FTO/Fto was knocked down by shRNAs or genetically knocked out. Via transcriptome-wide m6A-sequencingand RNA-sequencing (RNA-Seq) assays in MONOMAC-6 AML cells with or without overexpression or knockdown of FTO, we identified two functionally critical targets of FTO, ASB2 and RARA.Forced expression of wild-type FTO, but not mutant FTO, reduced expression of RARA and ASB2. Forced expression of either ASB2 or RARA largely recapitulated the phenotypes caused by FTO knockdown. Moreover, the effects of overexpression or knockdownof FTO can be largely rescued by that of RARA or ASB2, indicating that they are functional important targets of FTO. Forced expression and knockdown of FTO reduced and increased, respectively, the m6A levels on ASB2 and RARA mRNA transcripts,and shortened and prolonged, respectively, the half-life of ASB2 and RARAmRNA transcripts in AML cells.Importantly, FTO reduced luciferase activity ofASB23'UTR, RARA3'UTR or RARA5'UTR constructs with intact m6A sites, while mutations in the m6A sites abrogated the inhibition, demonstrating that FTO-mediated gene regulation relies on its demethylase activity. Upon ATRA treatment, FTO was significantly down-regulated, while RARA and ASB2were up-regulated in NB4 APL cells. Forced expression of FTO noticeably suppressed, while depletion of FTO enhanced, ATRA-induced cell differentiation.Forced expression of either RARA or ASB2 could also substantially enhance NB4 cell differentiation. Conclusions In summary, we provide compelling in vitro and in vivo evidence demonstrating that FTO, an m6A demethylase, plays a critical oncogenic role in cell transformation and leukemogenesis as well as in ATRA-mediated differentiation of leukemic cells, through reducing m6A levels in mRNA transcripts of its critical target genes such as ASB2 and RARA and thereby triggering corresponding signaling cascades. Our study highlights the functional importance of the m6A modification machinery in leukemia. Disclosures No relevant conflicts of interest to declare.

Abstract: Virus-specific T cells have proven highly effective for the treatment of severe and drug-refractory infections after hematopoietic stem cell transplant (HSCT). However, the efficacy of these cells is hindered by the use of glucocorticoids, often given to patients for the management of complications such as graft-versus-host disease. To address this limitation, we have developed a novel strategy for the rapid generation of good manufacturing practice (GMP)–grade glucocorticoid-resistant multivirus-specific T cells (VSTs) using clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated protein 9 (Cas9) gene-editing technology. We have shown that deleting the nuclear receptor subfamily 3 group C member 1 (NR3C1; the gene encoding for the glucocorticoid receptor) renders VSTs resistant to the lymphocytotoxic effect of glucocorticoids. NR3C1-knockout (KO) VSTs kill their targets and proliferate successfully in the presence of high doses of dexamethasone both in vitro and in vivo. Moreover, we developed a protocol for the rapid generation of GMP-grade NR3C1 KO VSTs with high on-target activity and minimal off-target editing. These genetically engineered VSTs promise to be a novel approach for the treatment of patients with life-threatening viral infections post-HSCT on glucocorticoid therapy.

Abstract: Immune checkpoint therapy has resulted in remarkable improvements in the outcome for certain cancers. To broaden the clinical impact of checkpoint targeting, we devised a strategy that couples targeting of the cytokine-inducible Src homology 2–containing (CIS) protein, a key negative regulator of interleukin 15 (IL-15) signaling, with fourth-generation “armored” chimeric antigen receptor (CAR) engineering of cord blood–derived natural killer (NK) cells. This combined strategy boosted NK cell effector function through enhancing the Akt/mTORC1 axis and c-MYC signaling, resulting in increased aerobic glycolysis. When tested in a lymphoma mouse model, this combined approach improved NK cell antitumor activity more than either alteration alone, eradicating lymphoma xenografts without signs of any measurable toxicity. We conclude that targeting a cytokine checkpoint further enhances the antitumor activity of IL-15–secreting armored CAR-NK cells by promoting their metabolic fitness and antitumor activity. This combined approach represents a promising milestone in the development of the next generation of NK cells for cancer immunotherapy.

Abstract: Cytomegalovirus reactivation and interleukin 15 are major contributors to NK cell repertoire diversity and maturation after CBT. An immature NK cell subset characterized by low diversity index and poor effector function was highly predictive of relapse after CBT.

Abstract: Introduction Serious infection is common in patients with multiple myeloma (MM), and associated with major morbidity and mortality. Immunoglobulin (Ig) replacement is frequently used to reduce frequency and severity of infections, however evidence is based on small trials conducted & gt;20 years ago. Ig utilisation and associated costs in this patient cohort are rising worldwide. In Australia (Aus), Ig for secondary hypogammaglobulinaemia is funded if total IgG is below lower limit of reference range with a history of recurrent or severe bacterial infection, or in severe hypogammaglobulinaemia (IgG & lt; 4 g/L excluding paraprotein). In New Zealand (NZ), patients generally require a history of recurrent infections and trial of oral antibiotics before commencing Ig. This study aims to outline Ig use in the "real-world" MM setting, identify variation in and predictors of use, and describe association of Ig with survival outcomes. Methods Retrospective review of patients registered on the MRDR, a prospective registry established in 2012, currently open at & gt;60 sites across Aus/NZ. Patients with a diagnosis of MM or plasma cell leukaemia with verifiable data regarding Ig use were included in this analysis. Ig use was confirmed using medical/laboratory records, national Aus Ig dispensing tool or the NZ Blood Service portal. Quality of survival outcomes and cause of death data was augmented via linkage with national death registries. Baseline patient/disease characteristics, therapy and survival outcomes were compared between patients who received any Ig within 24 months of MM diagnosis and non-recipients using chi-square tests for categorical variables and rank sum tests for continuous variables. Variation in Ig use across sites were compared. Kaplan-Meier survival analysis was used to estimate time to Ig and duration of use. A time-dependent Cox analysis was used to compare survival for patients whilst on and off Ig therapy. All statistical analysis was completed on STATA/IC statistical software v16.1 (College Station, TX, USA). Results As of July 2021, & gt;4600 patients with plasma cell dyscrasias are registered on the MRDR. Of these, 2025 patients from 13 sites (12 Aus across five states/territories and one NZ) with verifiable data on Ig were included. 242 (12.0%) received Ig during follow-up, with a median time from MM diagnosis to Ig use of 14.2 months, median duration of Ig use of 54.9 months, and median patient follow-up time of 23 months (Figure 1). At 24 months following MM diagnosis, 14.9% (160 of 1075 patients reaching 24-month follow up) received Ig, and a further 7.6% (82 patients) received Ig later, after 24 months. Administration of Ig within 24 months post-MM diagnosis varied widely across Aus states and territories (2.7%-29.6%), and was 2.0% from one NZ site. Patients administered Ig within 24 months post-MM diagnosis had lower baseline IgG (3.7 vs. 5.6 g/L, p & lt;0.001), IgA (0.3 vs. 0.5 g/L, p=0.027), IgM (0.20 vs. 0.21 g/L, p=0.019) and total serum Ig levels (28.0 vs 56.0 g/L, p & lt;0.001), were more likely to have abnormal Fluorescence in situ hybridization (FISH) results (83.5% vs. 62.7%, p & lt;0.001) and receive immunomodulatory drugs (IMiDs) (28% vs. 12.3%, p & lt;0.001) or anti-CD38 therapy (4.5% vs. 1.2%, p=0.004) first-line compared with patients not administered Ig (Tables 1, 2). Ig use was not associated with an overall survival benefit (HR=0.79, 0.50-1.24, p=0.3). At time of last follow-up, there were 577 deaths (28.5%). Available data on cause of death was available for 175 deaths (30.3%). Of these, 65 deaths (37.1%) had infection listed as primary/secondary cause of death (COD). 64 of 65 infection-related deaths occurred in non-Ig recipients. In patients who received Ig (at any time), 12.5% had infection as primary/secondary COD, compared to 38.5% in non-recipients (p=0.14). Conclusion This Aus/NZ analysis found that 14.9% of MM patients received Ig by 24 months post-MM diagnosis with wide variation in practice. Ig use was associated with lower baseline Ig levels (all subtypes), abnormal FISH and first-line IMiD/anti-CD38 therapy. In this "real-world" cohort, Ig use was not associated with survival, highlighting the need for well-designed contemporary studies to inform evidence-based patient selection, especially with increasing use of Ig and targeted anti-myeloma therapies, and a high burden of infection-related mortality. Figure 1 Figure 1. Disclosures Hamad: Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Harrison: Haemalogix: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Roche/Genentech: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Janssen Cilag: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene/ Juno/ BMS: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Abbvie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Eusa: Consultancy, Honoraria, Speakers Bureau; Sanofi: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; GSK: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Terumo BCT: Consultancy, Honoraria. Mollee: Janssen, Pfizer: Research Funding; Amgen, BMS, Janssen, Caelum, EUSA, Pfizer, SkylineDx, Takeda: Membership on an entity's Board of Directors or advisory committees, Other: No personal fees received. Quach: GlaxoSmithKline: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Antengene: Consultancy, Membership on an entity's Board of Directors or advisory committees; CSL: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen/Cilag: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; Sanofi: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Karyopharm: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Spencer: Celgene: Honoraria, Research Funding, Speakers Bureau; Janssen: Honoraria, Research Funding, Speakers Bureau; Amgen: Honoraria, Research Funding; Bristol Myers Squibb: Research Funding; Takeda: Honoraria, Research Funding, Speakers Bureau; STA: Honoraria. Wood: Amgen, Celgene, Gilead, Janssen, Novartis, Sanofi, Takeda: Research Funding; Abbvie, Amgen, Antengene, Bristol-Myers Squibb, Celgene, Gilead, GSK, Janssen, Novartis, Sanofi, and Takeda.: Other: The Australian and New Zealand Myeloma and Related Diseases Registry (MRDR) has received funding from Abbvie, Amgen, Antengene, Bristol-Myers Squibb, Celgene, Gilead, GSK, Janssen, Novartis, Sanofi, and Takeda. . McQuilten: Amgen, Celgene, Gilead, Janssen, Novartis, Sanofi, Takeda: Research Funding; Abbvie, Amgen, Antengene, Bristol-Myers Squibb, Celgene, Gilead, GSK, Janssen, Novartis, Sanofi, and Takeda.: Other: The Australian and New Zealand Myeloma and Related Diseases Registry (MRDR) has received funding from Abbvie, Amgen, Antengene, Bristol-Myers Squibb, Celgene, Gilead, GSK, Janssen, Novartis, Sanofi, and Takeda. .

Abstract: Introduction: A number of Clinical trials have demonstrated the feasibility, safety and efficacy of cell and gene therapy for cancer, autoimmune disorders and infectious disease. Strategies that enhance the function and survival of immune cells are critical for the success of immunotherapy. We have developed a strategy for the ex vivo expansion of off-the-shelf viral-specific T cells (VSRs) from healthy donor buffy coat which have been extremely effective in eradicating refractory cytomegalovirus (CMV), polyomavirus and adenovirus infections in immunocompromised patients. Glucocorticoids commonly used to treat graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation (HSCT) are a common cause of iatrogenically-induced immunosuppression and contribute the risk of life-threatening viral-infections. To render VSTs resistant to the lymphocytotoxic effect of glucocorticoids, we have developed a novel strategy to silence the expression of the glucocorticoid receptor using RNA-guided endonucleases CRISPR (clustered regularly interspaced short palindromic repeats) and CRISPR-associated (Cas) 9 gene editing.. Methods: The technique involves the expansion from donor blood of CMV, BKV or adenoviral-specific T cells using peptide libraries from the immunodominant viral proteins followed by CRISPR knockout of exon 2 of the GR gene on chromosome 5 of the human NR3C1 gene. Cells are electroporated with the RNP (Cas9 plus guide RNA) complex (IDT pre-designed alt-R crispr Cas9 platform) using Neon electroporation and the Amaxa 4-D nucleofector system. Results: GR knockout efficiency in ex vivo expanded virus-specific T cells was consistently 〉 90%. In vitro experiments confirmed the resistance of VSTs to corticosteroid treatment as assessed by annexin V assay. GR KO VSTs maintained potent antiviral activity as assessed by their ability to proliferate and release effector cytokines in response to viral antigens. Conclusions: CRISPR gene-editing to knock-out the glucocorticoid receptor gene in viral-specific T cells can preserve the activity of VSTs in the presence of corticosteroid-induced immunosuppression. Engineering runs using GMP-compliant Cas9 protein and gRNA are underway in anticipation of a clinical trial. Disclosures Champlin: Sanofi-Genzyme: Research Funding; Actinium: Consultancy; Johnson and Johnson: Consultancy.

Abstract: Approximately 10% of human acute leukemias are involved in chromosomal translocations between the mixed lineage leukemia (MLL) gene and over 50 partner genes. MLL-rearranged leukemias occur preferentially in infant and young children and are often associated with poor outcome. MicroRNAs (miRNAs) are an abundant class of small noncoding RNAs which repress gene expression and mRNA stability by base pairing with target mRNAs usually at the 3’-untranslated regions (UTRs). The ten-eleven translocation 1 (TET1), the founding member of the TET family of enzymes (TET1/2/3) that convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), was first identified in MLL-rearranged leukemia. But its definitive role in leukemia was unclear until our recent report published in PNAS (Huang H. et al. 2013). In contrast to the frequent repression and tumor-suppressor roles of the three TET genes observed in various cancers, we showed that TET1 is a direct target of MLL-fusion proteins and significantly up-regulated in MLL-rearranged leukemia, leading to a global increase of 5hmC level. Furthermore, Tet1 plays an indispensable oncogenic role in MLL-rearranged leukemia, through coordination with MLL-fusion proteins in regulating their critical co-targets including Hoxa/Meis1/Pbx3 genes. However, whether TET1 is also post-transcriptionally regulated by miRNAs in hematopoietic cells remains unknown. In the present report, through genome-wide miRNA expression profiling assays, we found that miR-26a and miR-29a were expressed at a significantly lower level in MLL-rearranged AML than in normal controls. The down-regulation of miR-26a and miR-29a is, at least in part, attributed to the transcriptional repression mediated by MLL-fusion proteins and MYC. Interestingly, both miR-26a and miR-29a target TET1 directly at the post-transcriptional level. More importantly, we showed that miR-26a or miR-29a significantly inhibited MLL-fusion-mediated cell transformation in vitro and leukemogenesis in vivo down regulating expression of Tet1 and its downstream target genes. Thus, our data suggest that the transcriptional repression of miR-26a and miR-29a is required for the aberrant overexpression and potent oncogenic role of TET1 in MLL-rearranged leukemia, and that miR-26a and miR-29a play important tumor-suppressor role in leukemogenesis. Taken together, our data reveals a previously unappreciated signaling pathway involving the MLL-fusion/Myc⊣miR-26a/miR-29a⊣Tet1 circuit in MLL-rearranged leukemia. Our data not only provides novel insight into our understanding of the complex molecular mechanisms underlying the pathogenesis of MLL-rearranged leukemia, but also may lead to the development of novel, more effective therapeutic strategies to treat this type of dismal disease. Disclosures No relevant conflicts of interest to declare.

Abstract: Acute myeloid leukemia (AML) is one of the most common and fatal forms of hematopoietic malignancies with diverse chromosomal and molecular abnormalities. The majority of AML patients do not survive more than 5 years. Advanced genomic studies reveal that both genetic and epigenetic abnormalities frequently occur in de novo AML. However, it remains a challenge to understand the complicated genetic/epigenetic regulatory networks and identify the functionally important nodes in these networks. There is an urgent need to develop effective therapeutic strategies based on these new insights. The ten-eleven translocation (Tet) proteins are important epigenetic regulators, which can convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) and lead to DNA demethylation. Among the three TET family members (TET1/2/3), TET2 was identified as a tumor suppressor in myeloid malignancies. Our lab recently reported that TET1 is highly expressed in MLL/KMT2A (Mixed Lineage Leukemia)-rearranged AML, a subtype of AML with poor prognosis. It is a direct target activated by MLL-fusions, and functions as an essential oncogene (Huang et al., PNAS, 2013). However, the function and regulatory pathway(s) of TET1 in AML remain poorly understood. MicroRNAs (miRNAs) are a class of small, non-coding RNAs that play important roles in posttranscriptional gene regulation. Dysregulation of miRNAs is frequently observed in AML. Results of our profiling assays show that miR-22 is widely down-regulated in all major subtypes of de novo AML (Jiang et al., Cancer Cell, 2012), implying a tumor suppressor function. However, an oncogenic role for miR-22 was recently reported in myelodysplastic syndromes (MDS) and breast cancer, in which TET2 was repressed by miR-22 as its direct target gene. Here we show that, amongst a group of miRNAs (e.g. miR-495 and miR-150, etc.) whose expression levels are repressed in AML, miR-22 exhibits the most potent and consistent inhibition on MLL-AF9-induced transformation of mouse bone marrow (BM) progenitor cells. Moreover, forced expression of miR-22 dramatically inhibits cell transformation and leukemogenesis induced by multiple fusion genes, such as MLL-fusions and RUNX1/AML1-ETO9a. Furthermore, the maintenance of various subtypes of AML (e.g., those induced by MLL-fusion, AML1-ETO9a or FLT3-ITD/NPM1c+) is also dependent on the repression of miR-22. Thus, our data demonstrate a potent tumor-suppressor role of miR-22 in AML. Surprisingly, our analysis of three (in-house and outside) large-scale AML datasets revealed that TET2 (and likely also TET3) expression levels exhibited a significant positive correlation, whereas only TET1 exhibited a significant negative correlation (r 〈 -0.32; p 〈 0.001), with miR-22 expression. Our subsequent ChIP/qPCR studies suggest an epigenetic repression on miR-22 transcription mediated by TET1 and its repressive cofactors such as SIN3A and EZH2, through their direct binding to the miR-22 promoter region and subsequent modifications of histone markers such as H3K27Me3 which, in turn, inhibit RNA polymerase II recruitment and, thereby, miR-22 transcription. Besides the TET1-mediated epigenetic repression, the miR-22 locus (within 17p13.3) is also affected by DNA copy loss in 8-20% of AML cases, further highlighting its tumor-suppressor role in AML. Further, through a series of data analyses followed by experimental validations and functional studies, we show that a set of critical oncogenes, including CRTC1, FLT3 and MYCBP, are functionally important direct target genes of miR-22 in AML and thus, miR-22 negatively regulates the CREB and MYC signaling pathways. Our proof-of-concept study shows that miR-22 RNA oligos formulated with dendritic nanoparticles significantly inhibit leukemia progression and extend the overall median survival of MLL-AF9-induced leukemic mice from 29 days to 54 days (n=10 per group, p 〈 0.001, log-rank test) in mouse BM transplantation assays, indicating the therapeutic potential of miR-22 in treating AML. Taken together, our results demonstrate a potent tumor-suppressor role of miR-22 in AML, and suggest the potential clinical application of miR-22-nanoparticles in treating AML. We also identified a TET1⊣miR-22⊣CREB/MYC regulatory pathway, which is critical in AML pathogenesis (see Fig. 1). Our findings also highlight potential distinct genetic/epigenetic mechanisms underlying de novo AML and MDS. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.