AbstractAcute Myeloid Leukemia Research Articles

Farmakogenomika i regulacija apoptoze u akutnoj mijeloidnoj leukemiji

Acute myeloid leukaemia (AML) is a heterogenous clonal hematopoietic malignancy primarily treated with combination of cytarabine (ara-C) and anthracyclines. Despite high remission rates, especially in younger patients, a vast majority of patients die due to relapse or chemotherapy/stem cell transplantation-related toxicity. The partial explanation for this grim clinical outcome lies in the patients' genetic variability. In this review, we will summarize how genetic polymorphisms of proteins, in metabolic paths of cytarabine and anthracyclines and proteins involved in regulation of apoptosis, influence efficacy and toxicity in the AML treatment.

Clofarabine Improves Relapse-Free Survival of Acute Myeloid Leukemia in Younger Adults with Micro-Complex Karyotype.

Acute myeloid leukemia (AML) encompasses heterogeneous entities with dismal outcomes. Intermediate and unfavorable-risk AML represent the most difficult-to-treat entities. We recently reported the benefit of the clofarabine-based consolidation (CLARA) regimen compared to the standard high-dose cytarabine (HDAC) regimen in younger AML patients. Here, we aimed at assessing the clinical significance of single-nucleotide polymorphism (SNP)-array alterations and their interactions with chemotherapy regimens. A SNP-array was successfully performed in 187 out of the 221 intent-to-treat patients (CLARA arm: n = 92 patients, HDAC arm: n = 95 patients). The CLARA regimen did not significantly improve relapse-free survival (RFS) among patients who displayed a complex karyotype when compared to the HDAC regimen (4-year RFS (4y-RFS): 36.4% vs. 18.8%, respectively; p = 0.134). Defining micro-complex karyotypes from at least four SNP-array lesions enabled us to refine and enlarge the subset of adverse patients. In such patients, the CLARA regimen significantly improved RFS compared to the HDAC regimen (4y-RFS: 44.4% vs. 13.8%, respectively; p = 0.004). From our study cohort, 8% of patients displayed TP53 mutations, which were associated with an impaired RFS (4y-RFS: 20.0% vs 43.7%; p = 0.029). In a multivariate analysis, micro-complex karyotypes remained the sole poor prognostic factor in the HDAC arm (hazard ratio (HR) = 2.324 (95% confidence interval (CI) = 1.337–4.041), p = 0.003). The SNP array represents a powerful and reproductive approach to refine adverse AML patients that may benefit from alternative consolidation regimens.

The face of remission induction.

Acute myeloid leukaemia (AML) is a heterogeneous disease in which prognosis is determined by cytogenetic and molecular aberrations as well as patient-related factors, including age, prior haematologic disorders, and comorbidities. Despite the diverse disease biology, the standard of care for remission induction therapy has changed very little since its inception in 1973. Next generation sequencing has helped to increase our knowledge of the disease pathogenesis, allowing us to develop targeted and possibly more effective treatment options. Seven new agents have been approved for the treatment of AML since 2017, all of which are directed toward a specific molecular subtype or patient population. With the advent of these therapies, a more optimal, patient-specific approach rather than the historical 'one-size fits all' model can be utilised. This review will discuss the role of these novel therapies in the remission induction setting.

The Prognostic Significance of PDE7B in Cytogenetically Normal Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is a malignant hematological disease in which nearly half have normal cytogenetics. We have tried to find some significant molecular markers for this part of the cytogenetic normal AML, which hopes to provide a benefit for the diagnosis, molecular typing and prognosis prediction of AML patients. In the present study, we calculated and compared the gene expression profiles of cytogenetically normal acute myeloid leukemia (CN-AML) patients in database of The Cancer Genome Atlas (TCGA), Gene Expression Omnibus (GEO) and dataset Vizome (a total of 632 CN-AML samples), and we have demonstrated a correlation between PDE7B gene and CN-AML. Then we proceeded to a survival analysis and prognostic risk analysis between the expression levels of PDE7B gene and CN-AML patients. The result showed that the event-free survival (EFS) and overall survival (OS) were significantly shorter in CN-AML patients with high PDE7B levels in each dataset. And we detected a significantly higher expression level of PDE7B in the leukemia stem cell (LSC) positive group. The Cox proportional hazards regression model showed that PDE7B is an independent risk predictor for CN-AML. All results indicate that PDE7B is an unfavorable prognostic factor for CN-AML.

The Mitochondrial Protease, Neurolysin (NLN), Regulates Respiratory Chain Complex and Supercomplex Formation and Is Necessary for AML Viability

Acute myeloid leukemia (AML) cells and stem cells have unique mitochondrial characteristics with an increased reliance on oxidative phosphorylation (OXPHOS). To identify new biological vulnerabilities in the mitochondrial proteome of AML cells, we conducted an shRNA screen and identified neurolysin (NLN), a zinc metalloprotease whose mitochondrial function is not well understood and whose role in AML has not been previously reported. To begin our investigation into the role of NLN in AML, we analyzed NLN gene expression in a database of 536 AML and 73 normal bone marrow samples. NLN was overexpressed in 41% of AML samples. Overexpression of NLN in primary AML cells compared to normal hematopoietic cells was confirmed by immunoblotting. To validate the results of the screen and to determine whether NLN is required for AML growth and viability, we knocked down NLN in the leukemia cell lines OCI-AML2, MV4-11, NB4, and TEX with shRNA. NLN knockdown reduced leukemia growth and viability by 50-70%. Moreover, knockdown of NLN in AML cells reduced the clonogenic growth of leukemic cells in vitro and the engraftment of AML cells into mouse marrow after five weeks by up to 80% and 85%, respectively. The mitochondrial function of NLN is largely unknown, so we identified NLN's mitochondrial protein interactors in T-REx HEK293 cells using proximity-dependent biotin labeling (BioID) coupled with mass spectrometry (MS). This screen identified 73 mitochondrial proteins that preferentially interacted with NLN and were enriched for functions including respiratory chain complex assembly, respiratory electron transport, and mitochondrion organization. Therefore, we assessed the effects of NLN knockdown on OXPHOS. NLN knockdown reduced basal and maximal oxygen consumption, but there were no changes in the levels of individual respiratory chain complex subunits. To understand how NLN influences OXPHOS, we examined the formation of respiratory chain supercomplexes (RCS). Respiratory chain complexes I, III, and IV assemble into higher order quaternary structures called RCS, which promote efficient oxidative metabolism. NLN knockdown significantly impaired RCS formation in T-REx HEK293, OCI-AML2, and NB4 cells, which was rescued by overexpressing wild-type shRNA-resistant NLN. RCS have not been previously studied in leukemia. Therefore, we analyzed their levels in primary AML patient samples and normal hematopoietic cells. RCS assembly was increased in a subset of AML patient samples and positively correlated with NLN protein expression (R2 = 0.83, p < 0.05), suggesting that NLN mediates RCS assembly in AML. To investigate how NLN may be regulating RCS assembly, we analyzed our BioID results to identify NLN interactors that are known regulators of supercomplex formation. Among the top interactors was the known RCS regulator, LETM1. Knockdown of NLN in AML cells impaired LETM1 assembly. Of note, knockdown of LETM1 also reduced growth and oxygen consumption of AML cells. As a chemical approach to evaluate the effects of NLN inhibition on AML cells, we used the allosteric NLN inhibitor R2, (3-[(2S)-1-[(3R)-3-(2-Chlorophenyl)-2-(2-fluorophenyl)pyrazolidin-1-yl]-1-oxopropan-2-yl]-1-(adamantan-2-yl)urea), whose anti-cancer effects have not been previously reported. R2 reduced viability of AML cells, as well as two primary AML culture models, 8227 and 130578. R2 impaired RCS formation in OCI-AML2, NB4, 8227, and primary AML cells. Moreover, R2 reduced the CD34+CD38- stem cell enriched population in 8227 cells, reduced LETM1 complex assembly, and impaired OXPHOS in OCI-AML2 and 8227 cells. Finally, we assessed the effects of inhibiting NLN in mice engrafted with primary AML and normal hematopoietic cells in vivo. Treatment of mice with R2 reduced the leukemic burden in these mice without toxicity. Moreover, inhibiting NLN targeted the AML stem cells as evidenced by reduced engraftment in secondary experiments. In contrast, inhibiting NLN did not reduce the engraftment of normal hematopoietic cells. Collectively, these results demonstrate that inhibition of NLN preferentially targets AML cells and stem cells as compared to normal hematopoietic cells. In summary, we defined a novel role for NLN in RCS formation. We show that RCS are necessary for oxidative metabolism in AML and highlight NLN inhibition as a potential therapeutic strategy. Disclosures Minden: Trillium Therapetuics: Other: licensing agreement. Chan:Agios: Honoraria; AbbVie Pharmaceuticals: Research Funding; Celgene: Honoraria, Research Funding. Schimmer:Medivir Pharmaceuticals: Research Funding; Novartis Pharmaceuticals: Consultancy; Jazz Pharmaceuticals: Consultancy; Otsuka Pharmaceuticals: Consultancy.

CRISPR-Cas9 Screen Identifies XPO7 As a Potential Therapeutic Target for TP53-Mutated AML

Acute myeloid leukemia (AML) is a devastating disease with a long-term survival rate of less than 30%. While AML is a genetically heterogeneous disease, TP53 mutation is among the most powerful risk factors in AML, underscoring the critical need to devise a novel therapeutic strategy for TP53-mutated AML. To identify genes/pathways whose loss are vulnerable to TP53 deficiency in AML cells, we performed genome-wide CRISPR-Cas9 screens using Trp53-knockout (KO) and wild-type (WT) mouse AML cells. To generate AML lines with a relatively "clean" genetic background, we established mouse AML lines harboring WT Trp53 with normal karyotype (Yamauchi et al. Cancer Cell 2018). We then generated Trp53-KO AML lines using a single guide RNA (sgRNA) targeting Trp53. Genome-wide CRISPR-Cas9 dropout screens were performed using these lines to identify genes/pathways whose loss are vulnerable to TP53 deficiency. Importantly, sgRNAs targeting Trp53 were enriched only in Trp53-WT AML cells, but not in Trp53-KO cells. Furthermore, sgRNAs targeting Mdm2, which encodes a E3 ubiquitin-protein ligase for Trp53 protein, were depleted only in Trp53-WT cells, attesting to the validity of our experimental system. We identified Xpo7, a putative nuclear/cytoplasmic transporter, as a factor necessary for the survival of Trp53-KO AML cells. sgRNAs targeting Xpo7 were enriched in Trp53-WT AML cells after the 16-day culture (Yamauchi et al. Cancer Cell 2018), while they were significantly depleted in Trp53-KO cells. Trp53-KO cells were vulnerable to Xpo7 depletion, while Xpo7 functioned as a Trp53-dependent tumor suppressor in Trp53-WT AML cells. As expected, either CRISPR/Cas9-mediated Xpo7 depletion or shRNA-mediated Xpo7 knockdown significantly delayed cell cycle progression and suppressed cell growth only in Trp53-KO AML cells, but not in WT cells. These findings were also validated using the DepMap (https://depmap.org), a publicly-available CRISPR/Cas9 dropout screen dataset. Significantly, XPO7 dependency was most correlated with that of TP53 and particularly evident in TP53-WT cell lines. We next performed CRISPR-Cas9 saturation mutagenesis scan targeting all Xpo7 exons using Trp53-WT and -KO mouse AML cells. Strikingly, sgRNAs targeting Xpo7 coding regions were significantly enriched only in the Trp53-WT cells, namely in the presence of an MDM2 inhibitor, supporting the notion that Xpo7 could function as a tumor suppressor when Trp53 function is intact and evident. In a stark contrast, the same sgRNAs, which target coding exons, were mostly depleted in Trp53-KO AMLs. Importantly, sgRNAs targeting the Xpo7 UTRs were unchanged/neutral regardless of experimental conditions (e.g. Trp53 status, MDM2 inhibitor treatment), serving as valid control for the experiments. Since Xpo7 presumably mediates the nuclear import and export of proteins, we hypothesized that Xpo7 promotes retention of Trp53 protein in the nucleus in Trp53-WT AML cells. As expected, Trp53 protein levels in the nucleus were significantly decreased, while those in the cytoplasm were increased upon Xpo7 depletion, revealed by Western blot. These data suggest that Xpo7 retains WT-Trp53 in the nucleus and functions as a Trp53-dependent tumor suppressor in Trp53-WT AML. Finally, to explore functional significance of XPO7 in human AML, we assessed correlation between XPO7 expression levels and AML subtypes and/or genetic background using publicly-available datasets. XPO7 mRNA levels were significantly upregulated in TP53-mutated AMLs in the TCGA datasets (Ley et al. NEJM 2013). XPO7 mRNAs levels were remarkably high in acute erythroid leukemia (AEL) cases, where TP53 mutations are frequently observed (Tyner et al. Nature 2018 and Iacobucci et al. Nat Genet. 2019). In fact, shRNA-mediated XPO7 knockdown significantly suppressed proliferation of HEL, a human AEL cell line harboring TP53 mutation. In summary, we identified a synthetic lethal relationship between TP53 and XPO7. Our study may facilitate the development of novel therapeutic strategies for TP53-mutated AML, such as AEL. Disclosures Akashi: Celgene, Kyowa Kirin, Astellas, Shionogi, Asahi Kasei, Chugai, Bristol-Myers Squibb: Research Funding; Sumitomo Dainippon, Kyowa Kirin: Consultancy.

Somatic Mutations Potentiating RAS-MAPK Signaling Confer Resistant Potential Against FLT3-Inhibitors to Acute Myelogenous Leukemia

Acute myeloid leukemia (AML) is one of the most common hematologic malignancies derived from a small number of self-renewing leukemic stem cells (LSCs). AML has various genetic abnormalities such as chromosomal aberration, single nucleotide variant and insertion/deletion. In particular, internal tandem duplication of FMS-like tyrosine kinase 3 (FLT3-ITD) is the most frequently found in ~ 25 % of AML cases, moreover, AML with FLT3-ITD is classified in unfavorable. FLT3 is fundamentally essential for survival of hematopoietic stem cells (HSCs) as well as LSCs, but once ITD is acquired, FLT3-ITD promotes the proliferation of AML by activating downstream enzymes. Various FLT3 inhibitors are now available as effective and promising drugs. On the other hand, the resistance against those inhibitors has been regarded as an emerging problem in clinics. To clarify the molecular machineries how AML cells acquire the resistance against FLT3 inhibitors, we compared the mutation status of paired AML samples consisted of diagnostic and relapsed samples. LSCs, which are quiescent and highly resistant against conventional chemotherapies, are considered to play a central role in recurrence. Since we and other group previously reported that TIM-3 clearly discriminates LSCs from residual normal HSCs, we purified and evaluated the mutations of CD34+CD38-TIM-3+ LSCs or CD34+ immature AML cells. The paired diagnostic and relapsed samples obtained from the identical 5 AML patient treated with Gilteritinib were subjected to whole exome sequencing (WES) analysis. CD3+ T cells were also purified from the diagnostic samples and used as a germ line control to detect somatic mutations acquired in AML cells. Libraries for WES were prepared using Sure Select XT HS and Human All Exon V6 Capture Kit in accordance with manufacturer's instructions. Then we performed WES using NextSeq 500 with 150bp pair-end read. Surprisingly, in all 5 cases examined, AML clones lost FLT3-ITD, whereas clones harboring mutations potentiating RAS-MAPK signaling became dominant at relapse: one case acquired the activated KRAS (Q61H) mutation and the remaining four cases newly acquired the previously-unidentified mutations in RAS-MAPK inhibitory genes. Such mutations in RAS-MAPK-inhibitory genes include DIRAS3 (W173X), RASA1 (E70G), RAP1GAP2 (K18X), RIN2 (S355fs), RASA4 (C655R), RASA4B (E141G), MAP2K7 (T66P) and PTPN7 (T14M). Of note, these genes belong to RAS-MAPK-inhibitory pathways and the encoded proteins are reported to suppress the activity of RAS-MAPK pathways. These RAS-MAPK-inhibitory mutations were exclusively detected in the relapsed AML clones, and contained stop-gain and frameshift mutations. We, therefore, hypothesized that RAS/MAPK activation driven by somatic mutations contributes to the acquisition of resistance against FLT3 inhibitors in LSCs. Consistent with such hypothesis, phosflow analysis revealed that basal ERK phosphorylation was retained or rather increased in the relapse CD34+ AML cells lacking FLT3-ITD, indicating that RAS-MAPK activation was maintained independent of FLT3-ITD. Furthermore, ASP2215 and AC220 inhibited ERK phosphorylation in the diagnostic CD34+ AML cells, whereas both inhibitors failed in the inhibition of ERK activation in the relapsed AML clones in all 3 cases analyzed. Gilteritinib also failed in inhibiting ERK in other relapsed AML cases, however, the combination of ASP2215 and MEK inhibitor efficiently suppressed ERK phosphorylation. All of relapse AML cases exhibited the emergence of AML clones harboring mutations associated with RAS-MAPK pathway genes, and therefore, the clinical sequence for detecting such mutations might be useful to assess the risk of FLT3-inhibitor treatment failure in clinics. Furthermore, our study showed the efficacy of MEK inhibitors to inhibit RAS-MAPK signaling activity even in the relapsed AML cells. Thus, the combination therapy consisted of FLT3-inhibitors and MEK inhibitors might be one of the promising therapeutic strategies to overcome FLT3-inhibitor resistance in AML. In summary, our study revealed that mutations potentiating RAS-MAPK pathway should play a central role in conferring the resistance against FLT3-inhibitors to AML. Disclosures Akashi: Sumitomo Dainippon, Kyowa Kirin: Consultancy; Celgene, Kyowa Kirin, Astellas, Shionogi, Asahi Kasei, Chugai, Bristol-Myers Squibb: Research Funding.

Differentiation Therapy with Novel Epigenetic Inhibitors in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is a malignant disease characterized by uncontrolled proliferation, differentiation arrest and accumulation of immature myeloid progenitors. Despite recent developments and the approval of new therapeutic agents in the last few years, long term survival of AML, particularly in elderly patients remains an unmet medical need.The use of all-trans retinoic acid (ATRA) in Acute Promyelocytic Leukemia has proven that differentiation therapy may significantly change the survival of AML patients, however the success in APL has not been translated to other groups of AML. Therefore, the identification of new therapeutic agents that may induce the differentiation of AML blasts represents an attractive new target. Furthermore, it is well known that epigenetic alterations have an important role in the development and maintenance of cancer and AML in particular. Thus, our aim was to develop new small molecules targeting epigenetic modifying enzymes like DNA methyltransferases (DNMT), histone methyltransferases or histone deacetylase (HDAC) with the aim of inducing differentiation in AML. We performed a screening of over 50 small molecules synthesized by our group. The design was performed in-house using a knowledge and structure based strategy and the read out of the screening was based on changes in expression of CD11b (a well described marker of myeloid differentiation) after in vitro treatment of AML cells lines. Interestingly, we found several compounds with high capacity to promote the differentiation of leukemic cells in AML cells lines at low non-cytotoxic doses, selecting CM-444 and CM-1758 as our lead compounds (Figure 1a).A complete biochemical characterization showed that both compounds are specific pan-HDACs inhibitors (HDACi). CM-444 and CM-1758 induced in vitro cell differentiation in all subtypes of AML, independently of the AML genetic subgroups or the presence of mutations, which was significantly more pronounced that differentiation induced by reference compounds such as Panobinostat, Vorinostat, Entinostat, Tubastatin or Quisinostat, previously described HDACi. CM-444 and CM-1758 also induced in vivo differentiation in xenogeneic models of AML. AML differentiation was associated with induction of CD11b, downregulation of c-MYC, overexpression of transcription factors that govern the myeloid differentiation and morphologic changes. In addition, these compounds promoted in vitro differentiation of patient-derived AML blasts. The complete transcriptome analysis by RNA-Seq carried out in AML cell lines after CM-444, CM-1758, Panobinostat or Vorinostat treatment showed changes in genes implicated in differentiation, but without explaining the differences among the different HDACi. Analysis of the complete acetylome and proteome before and after treatment with CM-444 and CM-1758 in comparison with other HDACi showed differential acetylation of non-histone proteins included in the GO categories of Zn binding proteins and nucleic acid binding proteins (Figure 1b). Most of these proteins are epigenetic enzymes and have been related to AML and myeloid differentiation, such as MLL2, EP300 or BRD4. In summary, we have developed and characterized novel epigenetic small molecules with a high in vitro and in vivo capacity of differentiating AML cells. These compounds might be an effective differentiation-based therapy to be tested in AML. Besides, the mechanism of differentiation of these compounds is due, at least in part, to the acetylation of non-histone epigenetic proteins, which are key in the myeloid differentiation. Disclosures Paiva: Celgene, Janssen, Sanofi and Takeda: Consultancy; Amgen, Bristol-Myers Squibb, Celgene, Janssen, Merck, Novartis, Roche and Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees. San-Miguel:Amgen, Bristol-Myers Squibb, Celgene, Janssen, MSD, Novartis, Roche, Sanofi, and Takeda: Consultancy, Honoraria.

Investigation of Homing Capacity As Prognostic Marker in Human Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is the most frequent myeloid malignancy in adults, causing death of about 90% of elderly patients. Cytogenetic and molecular abnormalities are used to categorize AML in favorable, intermediate and adverse molecular risk groups. Classification in one of these groups will directly affect clinical decision-making. Although molecular criteria have significantly improved prognostication and thus AML patient stratification and risk-based treatment, they still do not allow full risk prediction. Using a xenotransplantation model of human AML samples into NOD/SCID/IL2Rgnull(NSG) mice, we have previously shown that favorable risk AML engraft NSG mice after longer latency when compared to intermediate or adverse risk AML (Paczulla et al., 2017). This suggests that this mouse model accurately depicts important features of human AML. To further investigate the relationship between in vivo leukemia latency in NSG mice and AML outcome in patients, we have expanded our analyses to a cohort of n=23 favorable AML patient samples with diverse cytogenetic and molecular features. We hypothesize that the time-to-leukemia induction in mice correlates with the capacity of homing to the bone marrow (BM) as well as with the outcome in patients. AML cells were derived from peripheral blood mononuclear cells of patients, and depleted for CD19-positive B- as well as CD3-positive T-cells by MACS. Patient-derived AML blasts were freshly thawed before each transplantation and intra-femorally transplanted into previously irradiated 6-8 week-old female NSG (500.000 cells per mouse). Transplanted mice were monitored for engraftment at two time points post-transplantation (16 weeks and 6 months) via BM biopsy and detection of human hematopoietic markers (CD33 or CD34, depending on patients features) using flow cytometry. At signs of disease or when more than 80% leukemic blasts were detected in the BM biopsy, mice were euthanized and leukemic infiltration of BM, peripheral blood (PB) and organs investigated using multi-color flow cytometry. Homing capacity was investigated by intravenous transplantation of 1.000.000 CFSE-labeled leukemic blasts in non-irradiated 6-8 week-old male NSG mice. 16 hours later, mice were euthanized and BM and PB analyzed for detection of fluorescent leukemic cells. Interestingly, all transplanted favorable AML samples were able to engraft NSG mice within 54 weeks after transplantations, with very different time-to-leukemia induction between patients. Survival proportion at week 16 was 81.5%, 78.1% at week 26 (6 months) and dropped to 27.3 % at week 40 (9 months). From the BM punctures performed at week 16 (n=65 mice, n=23 AML patients), 44.62% showed more than 1%, 76.92% more than 0.1% and 84.62% more than 0.01 % human leukemic cells. Notably, although some mice showed very low engraftment at week 16, they nevertheless developed leukemia at a later time point. Interestingly, results from BM punctures seem to nicely correlate with long-term survival. Indeed, mice for which more than 1% engraftment was detected at the 16-week time-point showed a 50% survival rate at week 26, 42% at week 26 and 27% at week 40 (in comparison to respectively 92%, 80% and 61% survival for mice with <1% leukemic cells at the same time-point). Furthermore, homing capacity of leukemic cells also seems to correlate with long-term engraftment (n=56 mice, n=20 AML patients). In fact, we could observe a tendency for increased homing to the BM for the samples that resulted in more than 1% engraftment at week 16 in comparison to those with less than 1% (median homing 0.0049% vs. 0.0029%). Taken together, our recent work demonstrates in a large set of patient samples that - in contrast to several previous publications - human AML cells of favorable molecular risk indeed routinely engraft NSG mice. Furthermore this work points out to correlations between homing capacity, BM engraftment at week 16 and long-term survival and time-to-leukemia in NSG mice. Comparative studies using AML cells of intermediate and adverse molecular risk are underway. These findings provide proof-of-principle data for a prospective clinical study analyzing correlations between AML cell homing capacity in mice and outcome of disease in patients. Disclosures No relevant conflicts of interest to declare.

Targeting Salt-Inducible Kinase 3 As a Therapeutic Approach for Acute Myeloid Leukemia

Acute myeloid leukemia (AML) remains a devastating illness, with a clear need for the development of novel anti-leukemic therapy. CRISPR-Cas9 functional genomic screens have revealed hundreds of genetic vulnerabilities in AML cells, which can be leveraged to nominate clinically actionable targets. Our previous CRISPR screens in AML cell lines identified Salt-Inducible Kinase 3 (SIK3) as an essential gene for a subset of AML cells with high expression of the oncogenic transcription factor MEF2C, such as AMLs harboring Mixed Lineage Leukemia (MLL)-fusion proteins. Mechanistically, we have shown that the maintenance of MEF2C transcriptional output requires SIK3, which phosphorylates histone deacetylase 4 (HDAC4), a repressive cofactor of MEF2C, and thus prevents HDAC4 from nuclear entry and from suppressing MEF2C activity. SIKs 1-3 are serine/threonine kinases belonging to the adenosine monophosphate-activated protein kinase (AMPK) family. The physiological function of SIKs has been investigated in the regulation of gluconeogenesis, myogenesis and osteogenesis, yet the critical role of SIK3 in AML growth is just emerging. Thus, the evaluation of its translational potential in relevant pre-clinic models, such as patient-derived-xenografts (PDXs), is necessary. To do so, we have established a novel MLL-AF9 PDX model tractable for CRISPR genome editing via lentivirally expressing Cas9 and serial transplantation in immunodeficient mice. The PDX model maintains stable Cas9 activity; knockout of SIK3 hampered leukemia growth in vivo, demonstrating the utility of this model and the functional significance of SIK3 in AML. In parallel, we tested the anti-leukemic effect of the tool compound YKL-05-099, which inhibits the SIK kinase family and has a suitable bioavailability in mice. YKL-05-099 treatment inhibited the growth of AML cell lines in vitro via inducing cell cycle arrest and apoptosis, and AML cells with high MEF2C protein expression tended to be more sensitive to YKL-05-099. YKL-05-099 was well-tolerated by mice, and after daily administration for a month, no obvious toxicities or physiological abnormalities were observed. Normal hematopoiesis was also sustained, as indicated by complete blood counts and lineage flow cytometry analysis. By contrast, a two-week treatment regimen successfully attenuated disease progression in vivo and extended animal survival in both an MLL-AF9 mouse AML and PDX models. Importantly, we have confirmed the action of the compound is via on-target inhibition of the SIK3-HDAC4-MEF2C axis. Western-blot analysis revealed that YKL-05-099 treatment led to rapid dephosphorylation and nuclear accumulation of HDAC4 in AML cells. Accordingly, similar to SIK3 knockout, ChIP-seq analysis showed that YKL-05-099 selectively reduced histone H3 lysine 27 acetylation at a small number of genomic locations, which are enriched for MEF2C occupancy sites. This finding was supported by RNA-seq analysis as well, which showed that YKL-05-099 treatment suppressed a similar transcriptional program as observed following genetic targeting of SIK3 or MEF2C. Additionally, either overexpressing a SIK3 gatekeeper mutant or deleting HDAC4 in MLL-fusion AML cells can attenuate their sensitivity to YKL-05-099. High MEF2C expression in AML is associated with poor response to conventional chemotherapy. Our findings validate SIK3 inhibition as a possible therapeutic approach in MEF2C-positive AML and provide a rationale for developing improved drug-like inhibitors of SIK3, with enhanced specificity and bioactivity, for definitive pre-clinical investigation and for future studies in patients with AML. Disclosures Lin: Leukemia & Lymphoma Society: Research Funding. Tarumoto:Lauri Strauss Leukemia Foundation: Research Funding. Gray:Gatekeeper, Syros, Petra, C4, B2S and Soltego.: Equity Ownership; Novartis, Takeda, Astellas, Taiho, Janssen, Kinogen, Voronoi, Her2llc, Deerfield and Sanofi.: Equity Ownership, Research Funding. Vakoc:Boehringer-Ingelheim: Research Funding; KSQ Therapeutics: Consultancy. Stegmaier:Rigel Pharmaceuticals: Consultancy; Novartis: Research Funding.

Myeloid-Lineage Enhancers Drive Oncogene Synergy and Represent a Novel Therapeutic Target in CSF3R-CEBPA Mutant AML

Myeloid-Lineage Enhancers Drive Oncogene Synergy and Represent a Novel Therapeutic Target in CSF3R-CEBPA Mutant AML

DC Vaccination Induces Antigen Specific Immune Responses in AML Patients: A 1-Year Interim Assessment

Acute myeloid leukemia (AML) is a lethal hematological malignancy characterized by high rates of relapse (50%). Studies of immune responses in AML patients led to identification of two leukemia-associated antigens (Wilms' tumor protein 1 (WT1) and preferentially expressed antigen of melanoma (PRAME) for which cellular and humoral immune responses were detected. Based on expression profiles in AML and normal tissues, WT1 and PRAME were selected as target antigens for a DC vaccine, aiming to elicit adaptive and innate immune responses in AML patients. The primary objective of the trial (EudraCT No.: 2014-003520-44) is to evaluate safety and feasibility of active immunotherapy with antigen-loaded autologous DCs. The secondary objectives are to assess DC vaccination-induced T cell responses against WT1 and PRAME and to evaluate the clinical outcome over a period of 2 years or until disease progression/relapse. WT1-positive AML patients, with or without PRAME positivity were included, if they had a morphological remission with or without hematological recovery (CRi) after induction chemotherapy and were not eligible for allogeneic hematopoietic stem cell transplantation. DCs were administered intradermally (2.5x106 WT1 plus 2.5x106 PRAME DCs) on a weekly basis for 4 consecutive weeks, followed by administration on week 6 and then every 4 weeks until 2 years, tumor progression or patient withdrawal. This report focusses on the immune-monitoring data collected during the first year of treatment. Multiple parameters were evaluated, including WT1- and PRAME specific T cell responses in peripheral blood (IFN-g ELISPOT), the recurrence of WT1- and/or PRAME-positive AML blasts in bone marrow (qRT-PCR) and peripheral blood (flow cytometry), and occurrence of AML-specific mutations in bone marrow (NGS). DCs were assessed for expression of WT1 and PRAME and costimulatory molecules by flow cytometry, while secretion of IL10 and IL12 was evaluated by double-color ELISPOT. Twenty CR/CRi AML patients were vaccinated, and vaccination was well tolerated, and no signs of autoimmunity were observed. 12/20 patients remained stable throughout the first year of treatment, while 8/20 relapsed. 2/8 relapsed patients died due to progression of disease during the first year. In patients in stable remission, 7/12 patients had no detectable IFN-γ ELISPOT response in peripheral blood, 3/12 mounted a response and 2/12 were not evaluable due to high background production of IFN-γ. Positive IFN-γ ELISPOT responses were observed within 1-30 weeks of treatment and associated with a decrease over time in levels of WT1 (3/3) and/or PRAME mRNA (1/3) expression. Except for 2 patients who remained MRD-negative throughout the first year of treatment, ELISPOT-negative patients in remission (5/7) exhibited low, relatively stable levels of target antigens. Stable remissions were associated with one (3/12) or no (8/12) AML-related mutations while one patient was not evaluable. AML relapses were observed within 50 days in 4 patients, within 51-100 days in 1 patient, and after more than 150 days in 3 patients. Overall, 6/8 relapsing patients exhibited positive IFN-γ ELISPOT responses, while 2 patients relapsing in <50 days failed to respond and one patient relapsing on day 77 exhibited an IFN-γ response at baseline. In all other relapsing patients, IFN-γ ELISPOT responses were detected within 6-30 weeks after vaccination, in association with rapid and progressive increases in levels of WT1 and/or PRAME. AML-related gene mutations were detected in 6/8 relapses, while one patient was not evaluable, and one had no mutation; In 4 of the relapsing patients, 3 to 6 mutations were detected, while in 2/8 a single mutation was observed. In conclusion, IFN-γ responses to WT1 and/or PRAME were detected in peripheral blood of 75% (6/8) of relapsing patients, but only 25% (3/12) of the patients in remission. It is tempting to speculate that detection of IFN-γ response in peripheral blood is linked to the concomitant presence of AML blasts in the periphery of relapsing patients. Stable or decreasing levels of WT1 and/or PRAME mRNA in bone marrow of most patients in remission is compatible with the hypothesis that a local response against AML antigens may be ongoing, despite a negative IFN-γ response in peripheral blood. Monitoring of the patients currently in remission may shed some light on the role of DC vaccination on the prevention of AML recurrence. Disclosures Eckl: Medigene Immunotherapies GmbH: Employment. Raffegerst:Medigene Immunotherapies GmbH: Employment. Schnorfeil:Medigene Immunotherapies GmbH: Employment. Prinz-Schulz:Medigene Immunotherapies GmbH: Employment. Fingerhut:Medigene Immunotherapies GmbH: Employment. Bigalke:BioNTech Innovative Manufacturing Services GmbH: Employment. Pinkernell:Medigene AG: Employment. Schendel:Medigene AG: Employment, Equity Ownership, Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Tafuri:Medigene Immunotherapies GmbH: Employment.

A Computational Based Approach for Identification and Validation of Gene Mutations As Surrogate Markers of Gene Essentiality in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is a hematologic neoplasm characterized by a remarkable phenotypic and genomic heterogeneity. The recent characterization of genomic subtypes of AML based on large sequencing studies has provided the rationale for the development of targeted therapies based on the presence of specific genomic abnormalities. However, long term survival particularly in older patients remains a unmet medicalneed. Additionally, recent studies using RNA interference (RNAi) libraries have determined the existence of genes that are essential for the survival of multiple cancer cells. Understanding the effect of genomic alterations (mutations, deletions, translocations) on gene essentiality could favor the development of targeted therapies for specific subgroups of AML patients. However, current statistical methods such as the Benjamini-Hochberg (BH) procedure have shown limitations for controlling the false discovery rate (FDR) and have suboptimal sensitivity (recall of true positives) because the P-value correction does not include any prior information of individual tests. For this reason, in this study we developed a new large-scale statistical algorithm, which combine the RNAi libraries (more than 17.000 genes) data with mutational profiles, to identify gene essentialities associated with specific genomic mutations in order to explore this approach in AML. We adapted the Independent Hypothesis Weighting (IHW) procedure to the problem of identifying mutations as surrogate markers of gene essentiality, by using the gene mutation state in each cell line as prior information of a IHW problem. This approach was tested in 19 tumor subtypes, of the Cancer Cell Line Encyclopedia (CCLE) showing that it recalls new discoveries that cannot be identified with standard procedures in 17 out of 19 tumors, including the identification of up to 1,000 discoveries in tumor types in which BH recalls no discovery. These results demonstrated the accuracy of the IHW-based approach to identify gene mutations as surrogate markers of gene essentiality in the future. Once validated, we applied this computational model to the15 AMLcell lines of CCLE. The number of discoveries with an FDR of 20% increases from 2 (using the traditional BH correction), to 38 using our procedure, showing NRAS as the top mutation biomarker in the ranking. Interestingly, the algorithm identified one essential gene (NRAS) for NRAS mutated (NRAS-mut) and another essential gene (PTPN11) for NRAS wild type (NRAS-wt) AML cells, covering all samples of AMLs. To validate this hypothesis, we examined the effect of two different specific siRNAs for each gene (siPTPN11 and siNRAS) on cell proliferation of four AML cell lines: two lines with NRAS-mut (HL-60 and OCIAML3) and two with NRAS-wt (MV4-11 and HEL). Downregulation of NRAS expression significantly decreases the cell proliferation only in the 2 NRAS-mutated AML cell lines. Whereas the inhibition of PTPN11expression produced an equivalent effect, but specifically in the 2 NRAS-wt AML cell lines (Figure 1). These results confirmed our predictions and showed the essential role of NRAS or PTNPN11 in AML cell lines either with NRAS mutated or wild type, respectively. These results demonstrate that the application of our algorithm in the context of specific gene mutation not only may allow identification of directed therapies based on the mutation but can also define new gene essentialities amenable for targeted therapies providing new therapeutic strategies in patients with AML and potentially in other tumors. Disclosures Paiva: Amgen, Bristol-Myers Squibb, Celgene, Janssen, Merck, Novartis, Roche and Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene, Janssen, Sanofi and Takeda: Consultancy. San-Miguel:Amgen, Bristol-Myers Squibb, Celgene, Janssen, MSD, Novartis, Roche, Sanofi, and Takeda: Consultancy, Honoraria.

Combinatorial Genetics Uncovers Novel Targets for the Treatment of Npm1/Cohesin Mutated AML

Acute myeloid leukemia (AML) is a cancer that results in an accumulation of abnormal myeloid cells in the bone marrow. While some targeted therapies are currently in development, the high level of genetic heterogeneity and low 5-year survival rate of AML underscore the need to uncover further therapeutic targets. 30 somatic mutations are recurrent in adult patients, with the average patient harboring 5-15 mutations (1). It is unknown how each genetic lesion interacts with others to change the course of the disease. Determining this necessitates the use of combinatorial genetics, which has the power to uncover novel targets for the treatment of each patient's unique disease. The most common mutation found in adults with AML (25-30% of patients) is in the NPM1 gene, and is referred to as NPM1c (1,2). This mutation results in a mislocalization of the Npm1 protein exclusively to the cytoplasm. While NPM1c is considered to be a driver of AML, mice harboring this mutation only develop disease after a prolonged latency (18 months) and with incomplete penetrance, suggesting additional mutations are acquired during transformation (3,4). Approximately 50% of patients that have an NPM1 mutation also contain a heterozygous mutation in a cohesin complex gene (STAG2, SMC1A, SMC3, or RAD21), resulting in haploinsufficiency (1). Although cohesin mutations alone are insufficient for transformation to AML, enhanced self-renewal of HSPCs and significant changes in gene expression are observed in several different models (5,6,7). Given the clinical data, we hypothesized that cohesin mutations would cooperate with NPM1c to enhance transformation to AML through changes in gene expression. To address this hypothesis, we crossed the inducible NPM1cflox/+and SMC3flox/+mouse models. Mice harboring both mutations developed AML with increased penetrance compared to NPM1c/+ mice (65% vs. 55%). Enhanced self-renewal was also observed in double vs. single SMC3-/+ or NPM1c/+ HSPCs in vitro. RNA sequencing revealed that double mutant HSPCs exhibit deregulation of a unique set of genes compared to single mutant HSPCs. In our analysis, we observed the Rac1/2 guanine nucleotide exchange factor DOCK1 to be overexpressed in the double, but not the NPM1c/+single, cells. High expression of DOCK1 has been correlated with decreased overall and disease-free survival in AML patients (8). Therefore, we hypothesized that Dock1 may be an attractive therapeutic target in COHESINMut;NPM1c AML. We found that shRNA-induced knockdown of DOCK1 or the use of a small molecule Dock inhibitor decreased cell growth and enhanced apoptosis in SMC3-/+;NPM1c/+ double mutant leukemias. Likewise, inhibition of Rac1/2 reduced cell growth, enhanced apoptosis, and decreased self-renewal in double mutant leukemic cell lines, suggesting that Dock1 may drive leukemogenesis through Rac1/2 activation. Collectively, our data suggest that Dock1/Rac represents a unique target for the treatment of patients harboring the Npm1/Cohesin mutational combination. Our results provide validity to the concept that combinatorial genetics can be used to target the unique genetic landscape of an individual patient. Disclosures Vassiliou: Celgene: Research Funding; Oxstem Ltd: Consultancy; Kymab Ltd: Consultancy, Other: Minor Stockholder. Viny:Mission Bio: Other: Sponsored travel; Hematology News: Membership on an entity's Board of Directors or advisory committees. Levine:Amgen: Honoraria; Imago Biosciences: Membership on an entity's Board of Directors or advisory committees; Prelude Therapeutics: Research Funding; C4 Therapeutics: Membership on an entity's Board of Directors or advisory committees; Loxo: Membership on an entity's Board of Directors or advisory committees; Isoplexis: Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Research Funding; Roche: Consultancy, Research Funding; Novartis: Consultancy; Gilead: Consultancy; Lilly: Honoraria; Qiagen: Membership on an entity's Board of Directors or advisory committees.

High Expression of BCAT1 promotes Acute Myeloid Leukemia Progression and Sensitize to PARP Inhibitor

Acute myeloid leukemia (AML) is a highly heterogeneous disease with poor clinical prognosis, especially to cytogenetically normal AML(CN-AML)patients, which belong to moderate prognosis group. The clinical outcomes of them are not consistent. Therefore, further exploration of novel biomarkers and development of new anti-tumor drugs is urgently needed. Recently, accumulating evidence have emerged and demonstrated that branched-chain amino acids (BCAA) are essential for tumor growth and proliferation. Branched-chain aminotransferase 1(BCAT1), a BCAA metabolic enzyme, which correlates with cancer aggression, not only that, it has been reported that overexpression of BCAT1in leukemia cells decreased intracellular αKG levels, showed the characteristics of stem cells and displayed a phenotype like cases carrying IDH mutations. By limiting intracellular αKG, BCAT1high expression AML cells prone to cause an increase in DNA damage probably via suppressing homologous recombination. We firstly analyzed the prognostic significance of BCAT1expression in CN-AML patients. Collectively, we found that patients with high levels of BCAT1had poor prognosis, the median overall survival time is shorter in BCAT1high group (356 days versus 570 days)(Figure A). Next, we performed gene knockout in THP-1 and MV4-11 cells with relatively high expression of BCAT1and gene overexpressing in the relatively low expression cell line OCI-AML3. In summary, BCAT1overexpression contributed to cell growth, whereas suppression of BCAT1markedly limited cell proliferation and colony-forming ability by blocking cell circle at G0/G1 phase(Figure B and C).The mice model we conducted further validated the role of BCAT1expression in vitro. Notably, we found that knockout and overexpression of BCAT1can respectively reduce and enhance the tumor burden(Figure D)and effects the time of survival(Figure E). Because both BCAT1high expression and IDH mutation could decrease αKG levels and suppress αKG dependent dioxygenases, it is tempting to speculate that there is a possibility of a "BRCAness" phenotype when BCAT1is overexpressed.We indeed observed a rise in the base line level of DNA damage marker γ-H2AX in BCAT1overexpressing cell line(Figure F). In addition, the sensitivity of cells to cisplatin is also positively correlated with BCAT1expression levels, the IC50 of BCAT1overexpressing cell line OCI-AML3 is much lower than control. Consistently, knocking out of BCAT1decreased sensitivity to cisplatin. So we further tested the relationship between PARPi talazoparib(BMN 673) and BCAT1expression, Use a gain of function approach we increased susceptibility to PARPi, together with the loss of function data, these results strongly suggest that BCAT1promotes cell sensitivity to PARPi(Figure G). We believe that the combination of high expression of BCAT1and PARPi produces synthetic lethality. In addition, the ability to trap PARP1 of PARPi may produce unacceptable toxicity when combined with conventional doses of cytotoxic chemotherapies. So, we next combined PARP inhibitors with DNA-damaging agent daunorubicin, the synergistic effect is promising on BCAT1overexpressed OCI-AML3 cell especially at high dose, however, the synergistic effect of the control group is weak, sometimes even manifested as antagonistic.Consistent with previous reports, IDH1/2mutAML is vulnerable to PARP inhibition as monotherapy, but especially when combined with daunorubicin treatment. These results also confirmed our hypothesis that BCAT1overexpression mimics the phenotype of IDHmutcells, not only as DNA hypermethylation, but also leads to increased DNA damage levels in cells, increasing sensitivity to PARP inhibitor and DNA-damaging drugs. Through qPCR and WB assays, we found the same trend: DNA damage response-related protein ATM was down-regulated after BCAT1overexpression, which preliminarily explained why DNA damage was increased after BCAT1overexpression. After exogenous supplementation with the aKG analog DM-aKG, the enhancement of DNA damage caused by overexpression of BCAT1can be reversed. In summary, this study indicated that CN-AML patients with high BCAT1expression had poor prognosis. BCAT1plays the role as oncogene, can induce DNA damage that renders AML cells sensitive to PARP inhibition and DNA damage agents, and can be used as a novel therapeutic option for AML. Figure Disclosures No relevant conflicts of interest to declare.

Enasidenib Drives Maturation of Human Erythroid Precursors Independently of IDH2

Acute Myeloid Leukemia (AML) remains one of the most difficult cancers to treat, with a 30% 2-year survival rate. High-throughput sequencing of AML patients has identified mutations, including FLT3, IDH1, and IDH2, for which targeted therapies have been developed. Enasidenib is an FDA-approved, first-in-class agent that preferentially inhibits IDH2-mutant activity and reduces levels of the oncometabolite 2-HG, inducing differentiation of IDH2-mutated blasts. Interestingly, greater than 50% of enasidenib-treated patients who had no objective clinical response still demonstrated improvement in their peripheral blood counts and reached RBC transfusion independence. The mechanism underlying this phenomenon is unknown but is of great clinical relevance given the high transfusion dependence and anemia-associated complications universally associated with AML. Thus, we sought to investigate how enasidenib drives normal hematopoiesis to improve quality of life and reduce morbidity in AML patients. In this study, we demonstrate that enasidenib enhances erythropoiesis from normal CD34+ hematopoietic stem and progenitor cells (HSPCs) derived from cord blood (CB) and bone marrow. Enasidenib doubled the proportion and total number of mature CD71+/GPA+ erythroblasts after 8 days of culture with EPO, SCF, and IL-3. In the presence of EPO, enasidenib induced a gene signature characteristic of maturing erythrocytes, with increased expression of GATA1 (1.3 fold), EPOR (2 fold), and KLF1 (1.4 fold), and decreased PU.1 (0.5 fold) and GATA2 (0.7 fold). Enasidenib-treated progenitor cells further demonstrated increased hemoglobin production (1.9 fold) and morphologic characteristics of increased erythroid maturation. Next, we sought to determine if enasidenib augments erythroid differentiation through IDH2 and IDH2-dependent pathways. First, we found that other IDH inhibitors (AG-120, AGI-6780, and AG-881) did not increase erythropoiesis at doses ranging from 1-10μM. As expected for normal HSPCs, 2-HG was not present at detectable levels in either the DMSO or enasidenib-treated conditions, and addition of 2-HG (50, 200μM) did not affect the ability of enasidenib to increase the proportion of CD71+GPA+ cells. Because it is possible that enasidenib acts through inhibition of wild-type IDH2, we generated CRISPR-Cas9 engineered IDH2 knockout (KO) CD34+ cells and treated them with enasidenib. Similar to wildtype cells, IDH2 KO CB CD34+ cells demonstrated a 3.4-fold increase in %CD71+GPA+ erythroid cells. Thus, enasidenib augments erythropoiesis independently of both mutant and wildtype IDH2 pathways. We then investigated the progenitor population that enasidenib acts on to drive erythroid maturation. Enasidenib did not increase the number of BFU-E or CFU-E colonies or the proportion of BFU-E (IL3R-CD34+CD36-) and CFU-E (IL3R-CD34-CD36+) progenitors in colony forming or liquid culture assays, respectively, leading us to conclude that enasidenib acts on more mature erythroid progenitors. Indeed, treating sorted mature CD71+ erythroid progenitors with enasidenib increased %CD71+GPA+ cells compared to DMSO control, whereas enasidenib treatment of CD71- early erythroid progenitors showed no effect. These observations provide evidence that enasidenib acts on CD71+ erythroid progenitors to increase late-stage erythroid differentiation. Given that CD71 allows for iron uptake into erythroid progenitors, we hypothesized that enasidenib modulates the heme biosynthesis pathway. Enasidenib inhibited the ABCG2 transporter, which effluxes protoporphyrin IX (PPIX), the direct precursor to heme, from the mitochondria and cytosol. Inhibition of ABCG2 by enasidenib could lead to PPIX accumulation within the cell, driving increased heme synthesis. To investigate this hypothesis, we treated cells with 20μM Ko143, a potent ABCG2 inhibitor, and observed a similar increase in %CD71+GPA+ cells as seen with enasidenib. Measurement of PPIX autofluorescence by flow cytometry and microscopy revealed an increase of PPIX in enasidenib-treated cells by 1.2-fold. Together, our data suggests that enasidenib drives maturation of CD71+ erythroid precursors independently of wildtype or mutant IDH2. Our results position enasidenib as a promising therapy to stimulate erythropoiesis and provide the basis for a clinical trial using enasidenib to improve anemia in a wide array of clinical contexts. Disclosures Majeti: Forty Seven Inc.: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; BioMarin: Consultancy.

Single Cell Transcriptomic Analysis of the Histone H3 K27M Mutation in Pre-Leukemic Hematopoietic Stem Cells

Acute myeloid leukemia (AML) develops in a step-wise manner from pre-leukemic clonal expansion to full-blown disease driven by aberrant epigenetic changes. Indeed, regulators of the epigenome such as DNMT3A, TET2, IDH1/2, EZH2 and ASXL1 are often mutated in pre-leukemia and myeloid malignancies. We and others identified K27M/I mutations in histone H3 in AML (Boileau et al. Nat Commun, 2019; Lehnertz et al. Blood, 2017). We demonstrated that K27 mutations are found in pre-leukemic hematopoietic stem cells (HSCs), are enriched in secondary AML, expand the functional human HSC pool and increase leukemic aggressiveness. Transcriptomic and epigenomic analysis determined that K27 mutations alter gene expression through a global decrease in promoter H3K27 tri-methylation and a gene-specific increase in H3K27 acetylation in leukemic cells (Boileau et al. Nat Commun, 2019). Here, we have analyzed the effects of the K27M mutation on HSCs at the single-cell level to understand its role in pre-leukemic clonal expansion. Healthy CD34+CD38- human cord blood cells were transduced with HIST1H3H WT or K27M and injected intrafemorally into sub-lethally irradiated NSG mice. After 14 weeks, bone marrow cells from the femur were collected and sorted for CD34+ transduced (GFP+) cells. Single-cell transcriptomics were performed by generating gene expression libraries from ~8,000 CD34+ cells using the 10X Genomics technology and sequenced using HiSeq4000. We have performed initial clustering and dimensionality reduction (t-SNE and UMAP) and identified 10 and 11 distinct clusters in the WT and K27M samples, respectively. Gene sets distinguishing the individual clusters have been determined. Using published gene lists for primitive hematopoietic cell types, the clusters have been assigned to specific cell types such as HSC, granulocyte-monocyte progenitors (GMP), common myeloid progenitors (CMP), multi-lymphoid progenitors (MLP) and megakaryocyte-erythroid progenitors (MEP) (Laurenti et al. Nat Immunol, 2013). Preliminary joint clustering analysis indicates the presence of two distinct clusters for the WT and K27M samples that were both assigned as "HSCs" in individual clustering. Further analysis to identify the differences in the clusters and cell populations between WT and K27M samples is being performed and will be presented at this meeting. Overall, this single-cell transcriptomic analysis will aid in determining the mechanism of action of the K27M mutant histone in pre-leukemic HSC clonal expansion. In addition, we will be performing similar single-cell analysis on HSCs expressing mutant ASXL1 as a comparison. Further understanding of the role of mutations in epigenetic regulators, such as histone H3 and ASXL1, in pre-leukemic clonal hematopoiesis will provide valuable insight on how to better prevent and treat AML and other myeloid malignancies. Disclosures No relevant conflicts of interest to declare.

HOXA11 Regulates Chemoresistance By Modulating p53 Gene Expression in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) exhibits large intrinsic variation in drug responsiveness due to its inherent heterogeneity. Therefore, it is important to understand the resistant mechanism in order to improve the treatment. In our previously study, the OCI-AML2-resistant cell lines were established to resist cytarabine (Ara-C) in the concentration of 50 µM (OCI-AML2 R50). The RNA-seq results showed that many genes changed in the resistant cells compared to wild type OCI-AML2 cells. One of the most remarkably decreased gene in resistant cells was HOXA11 (Homeobox A11). It is the part of the A cluster on chromosome 7 and encodes a DNA-binding transcription factor which regulates gene expression, morphogenesis, and differentiation. In this study, we have evaluated the importance of HOXA11 in AML chemoresistance. We found that knockdown of HOXA11 repressed the WT OCI-AML2 cell proliferation and increased the population of cells expressing CD123 and CD47 LSC (Leukemia stem cell) markers and enhanced the resistance to Ara-C in vitro, while overexpression of HOXA11 showed the reverse effect. These results support the idea that HOXA11 promotes drug sensitivity and apoptosis in AML. However, the result also showed that overexpression of HOXA11 repressed the OCI-AML2 R50 cell proliferation and enhanced the resistance. Therefore, HOXA11 plays opposite role in sensitive cells and resistant cells. We further investigated the mechanism for these effects. We found that knockdown of HOXA11 decreased the p53 gene expression and overexpression of HOXA11 increased the expression of p53 in OCI-AML2 and R50 cells. Further, in OCI-AML2 R50 cells p53 has a hotspot mutation in DNA binding site and studies have shown that p53 mutation enhance cancer cell survival and chemoresistance. Therefore, our study shows dual roles for HOXA11 in cell survival. In p53 wild type parental AML2 cells, HOXA11 induces wild type p53 expression to enhance drug sensitivity while in resistant cell, HOXA11 promotes mutant p53 expression and enhances the resistance of chemotherapy. Disclosures No relevant conflicts of interest to declare.

Combined Thalidomide and Recombinant Human Interferon-α-1b and Interleukin-2 for Acute Myeloid Leukemia of Various Disease Status: A Multi-Center Prospective Study

AML (acute myeloid leukemia) patients with relapsed refractory diseases or in poor physical conditions have very limited choices of appropriate treatment regimens, and most alternative options are costly. Here, we reported that an affordable regimen with combined recombinant human interferon-α-1b (IFN-α1b), thalidomide, and recombinant interleukin-2 (IL-2) (the ITI regimen) achieved varying degrees of therapeutic effects in AML patients of various disease status and vulnerabilities. ITI regimen was administrated as follow: a subcutaneous injection of IFN-α1b 60 μg qod, IL-2 1 million unit qod, and 200mg thalidomide tablet orally taken every night before sleep. Group A Sixty-eight patients who were with relapsed or refractory AML were enrolled, sixty finished at least one course, a response rate (CR+CRi) of 16.7% (10/60) was observed, and, and 7 (11.7%) patients achieved partial remission.(Table 1) Group B Eighteen patients with morphologically complete remission and consistently positive MRD were enrolled, each patients underwent ITI regimen for 2 months at least. According to the criteria of the WHO risk stratification, 7 were with a favorable risk, 8 were with a intermediate risk, and 3 were with a high risk. Patients with FLT3-ITD mutations were treated with oral sorafenib during early induction and consolidation treatment. These 18 patients received conventional dosage of ITI regimen. Seven patients had a negative MRD after 1 or 2 months. The MRD levels of 3 patients significantly decreased after one or two months. Five patients suffered a Morphological relapse. Another 3 patients failed to the conventional dosage of ITI regimen with an increased MRD rate, but it decreased after we made a modification of IFN-α1b and IL-2 administration daily. The response rate of the 18 patients in Group B was 72.2%, where MRD < 0.01% was defined as the negative threshold. (Table 2) Group C Eighty-eight patients with MRD-negative AML after consolidation were enrolled. Among the 88 patients with initial CR, 11 (12.5%) relapsed during the maintenance period (Figure 1). All these 11 patients relapsed within 2 years, with a median recurrence period of 20 months, of which 2, 4, and 5 patients, respectively, were with favorable, intermediate, and high risk. The indicated that this regimen effectively reduced the relapse rate.(Table 3) Disclosures No relevant conflicts of interest to declare.

Assessing Metabolic Intervention with a Glutaminase Inhibitor in Real-Time by Hyperpolarized Magnetic Resonance in Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is an aggressive hematopoietic disease characterized by glutamine-dependent metabolism. A novel glutaminase (GLS) inhibitor, CB-839, is currently under evaluation for treatment of hematopoietic malignancies and solid tumors. Our purpose was to measure cellular changes in AML associated with CB-839 treatment and to test the ability of hyperpolarized pyruvate for interrogating these changes to OCI-AML3 cells. Our results show that treatment with CB-839 interfered with the citric acid cycle, reduced the NADH/NAD+ ratio and ATP levels, reduced cell proliferation and viability, and reduced the basal and maximal respiratory capacities [oxygen consumption rate (OCR)]. We observed a reduction of the conversion of hyperpolarized pyruvate to lactate in cell lines and in a mouse AML model after CB-839 treatment. Our in vitro and in vivo results support the hypothesis that, in AML, glutamine is utilized to generate reducing equivalents (NADH, FADH2) through the citric acid cycle and that reduction in redox state by GLS inhibition decreases the rate of pyruvate to lactate conversion catalyzed by lactate dehydrogenase. We propose hyperpolarized pyruvate/lactate measurement as a method for direct monitoring of metabolic changes occurring in AML patients receiving CB-839. With further optimization, this method may provide a noninvasive imaging tool to assess the early efficacy of therapeutic intervention with GLS inhibitors.