AML Samples Research Articles

Anti-Leukemic Activity of STRO-002 a Novel Folate Receptor-α (FR-α)-Targeting ADC in Relapsed/Refractory CBF2AT3-GLIS2 AML

BACKGROUND: CBF2AT3-GLIS2 (CBF/GLIS) oncogenic fusion is the underlying genomic cause of "RAM" phenotype AML, a megakaryoblastic subtype exclusively seen in infants and young children. CBF/GLIS AML is a highly refractory and uniformly fatal subtype of AML with limited response to conventional chemotherapy. We have demonstrated that FR-α, a known target in ovarian cancer, is expressed in CBF/GLIS AML cells and this expression is causally linked to the fusion oncogene. STRO-002 is currently under clinical investigation for the treatment of relapsed/refractory ovarian and endometrial cancer (NCT03748186). Preclinical studies have tested and confirmed the potent in vitro and in vivo anti-leukemia activity of STRO-002 in CBF/GLIS AML cell lines and xenograft models (Le et al. ASH 2021). Based on these compelling preclinical pharmacology observations, Sutro Biopharma evaluated patients on a case-by-case basis for consideration of a single patient IND (compassionate use) program for pediatric patients with CBF/GLIS AML. METHODS: Key criteria for consideration of STRO-002 were CBF2AT3-GLIS2 oncogenic fusion and FR-α expression by flow cytometry in AML sample. Key exclusion criteria were co-existent acute/uncontrolled infection and compromised organ function (pulmonary, hepatic, renal, etc). Between August 2021 and July 2022, 16 patients received STRO-002. Median age at treatment was 2 years (range 6 months-7 years). Median number of prior therapies was 3 (range 1-8). Eight patients had relapsed after stem cell transplant (SCT) and the remaining 8 had primary refractory (induction failure) or relapsed AML. RESULTS: In the 16 patients treated, 10 received STRO-002 as monotherapy, and 6 received combination with chemotherapy (fludarabine/cytarabine, decitabine, methotrexate (MTX), or dasatinib). All 16 pts were evaluable for response, best observed response included 7 CRs with 6 MRD negative remissions. Of these CRs, 3 occurred after monotherapy and 4 with combination therapy: MTX/fludarabine/cytarabine (n = 1), decitabine/DLI (n = 1), fludarabine/cytarabine/G-CSF (n = 1) and dasatinib/DLI (n = 1). The remaining 9 patients had stable disease or transient decrease in disease burden. Of the 7 patients who achieved a CR, 4 proceeded to SCT, 2 are receiving DLI and 1 will receive consolidation therapy. Ten (63%) of the 16 evaluable patients are alive and the other 6 died of progressive disease. Follow-up is limited for the surviving patients. Of note were 6 patients who achieved an MRD negative CR, two with primary refractory disease, and four who had relapsed after an allogeneic SCT. These six patients with significant disease had deep response to STRO-002 with MRD negative remission. One patient had a rapid decline in marrow blasts and had no evidence of disease by flow after 3 single agent every other week (QOW) doses of STRO-002. A second patient had a similarly rapid response to single agent STRO-002 with clearance of all detectable disease after a second QOW dose of STRO-002. ANC for both patients one week after last dose of STRO-002 was >1000. These patients were consolidated with allogeneic stem cell transplant or with DLI. STRO-002 was generally well tolerated. Collection and review of treatment-emergent AEs (TEAEs) is ongoing. Treatment-related events ≥ grade 3 included neutrophil count decreased (n = 3), platelet count decreased (n = 3), anemia (n = 2), WBC decreased (n = 2), hyperbilirubinemia (n = 1), bacteremia (n = 1), febrile neutropenia (n = 1), urticaria (n = 1), generalized edema (n = 1), aspartate aminotransferase elevation (n = 1), alanine aminotransferase elevation (n = 1). CONCLUSIONS: STRO-002 has promising activity in relapsed/refractory CBF2AT3-GLIS2 AML, a disease that tends to be highly refractory to all standard-of-care (SOC) therapies. Further, STRO-002 is well tolerated as a monotherapy agent and in combination with SOC therapies. Patients with low tumor burden were more likely to achieve CR and transition to SCT, DLI and CAR-T. Deep molecular and flow cytometric remission was achieved in a cohort of patients. Patients with primary refractory disease (no prior transplant), 1-3 prior lines of therapy, low disease burden, or without extramedullary disease appeared to have a more potent response to STRO-002. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Targeting of SFK and G6PD Contributes to Anti-Leukemic Activity of Novel FLT3 Trisubstituted Purine Inhibitors: Effective Therapeutic Approach for AML with FLT3 Mutations

We have recently developed compound LGR3922, a potent inhibitor of FLT3 receptor tyrosine kinase, which displays selectivity and potency in in vitro and in vivo AML models (compound 7d, Gucky T et al, J Med Chem. 2018;61(9):3855). In addition, LGR3922 showed strong inhibitory activity against Src family kinases (SFK) in the in vitro settings (Fig. 1A; ASH meeting abstract, Radosova Muchova et al, Blood. 2016;128(22):1569). Here, we present additional characterization of the lead compound LGR3922 and demonstrate its potent antileukemic activity in MLL/FLT3-ITD cell lines, murine xenografts and ex vivo primary AML samples. LGR3922 exhibited strong anti-proliferative effect against MV4-11 and MOLM-13 cells. Contrary to quizartinib (quiz), LGR3922 simultaneously reduced phosphorylation levels of FLT3-SFK and their downstream molecules STAT5 and ERK in both AML cell lines. Interestingly, the inhibitory effect of LGR3922 on SFK, STAT5 and ERK increased over time, especially in the MOLM-13 cells. Similar results were only seen for the combination treatment with quiz and dasatinib (das). Dual-targeting of FLT3-SFK by LGR3922 was further confirmed on MV4-11 xenografts. In the long-term in vivo experiment with repeated doses of the tested inhibitors, LGR3922 showed similar trends in initial treatment response as the combination treatment with quiz and das; quiz exhibited less efficient inhibitory effects at this stage. The tumor reduction was most significant (p ≤ 0.0001) in LGR3922 group; 3 out of 6 tumors were completely eradicated during the treatment period (Fig. 1B). Complete tumor eradication (in 1 out of 5 animals) over the treatment period was then seen only in combination quiz and das (Fig. 1B). No adverse effects on blood count parameters or body weight were associated with LGR3922 compared to combination treatment with quiz and das, where temporary body weight loss and only limited weight gain were observed. Next, we searched for additional target of the FLT3/SFK inhibitor that would have a synthetic lethal effect for the kinase pair. Besides the kinases that belong to other druggable nodes of AML-associated networks (Fig. 1A), we also tested antioxidant defense enzymes, previously validated as being synthetic lethal with FLT3 inhibitor therapy (Gregory MA et al. Proc Natl Acad Sci U S A. 2016;113(43):E6669). Indeed, we observed that LGR3922 inhibited G6PD activity, already after one-hour exposure (with a dose response over the concentration range), in contrast to quiz and das. We hypothesize that this activity modulating leukemia cell redox metabolism, cooperating with FLT3/SFK inhibition, may elicit more effective eradication of AML cells than quiz and das or quiz/das combination. We then evaluated toxicity of LGR3922 on normal hematopoiesis using colony forming unit (CFU) assays with progenitor cells from several healthy donor controls. LGR3922 inhibited colony formation in a dose-dependent manner; maximal tolerated concentration (MTC) for all healthy samples was 100 nM, in some samples the reduced granulocyte-macrophage (CFU-GM) and burst forming unit-erythroid (BFU-E) colony forming ability was not observed up to a concentration of 200 nM. Quiz and das exposure showed MTCs at 20 nM and 5 nM, respectively; in quiz/das combination at 10 nM/0.5 nM; 10 nM/5 nM was already toxic. Then, we performed CFU assays on 4 primary human AML samples with FLT3-ITD mutations (n=3) or FLT3-TKD+KMT2A::MLLT3 (n=1). Cultures with LGR3922 at 100 nM reduced the number of colonies by ∼70% in all FLT3-ITD patient samples. The inhibitory effects were comparable to or a little more potent than MTCs of quiz or quiz/das combination. Das culture had significantly lower effect on colony formation for the tested concentrations. In the FLT3-TKD+KMT2A::MLLT3 positive sample, fusion transcript was quantified in harvested colonies. While LGR3922 significantly reduced KMT2A::MLLT3 fusion transcript, no effects were observed for MTCs of single agent cultures with quiz or das. Only a combination of quiz/das exceeding MTC (10 nM/5 nM) resulted in a slight decrease of KMT2A::MLLT3 fusion transcript levels. Taken together, these data suggest effectiveness of our new LGR3922 compound and provide a rationale for further development and testing of this molecule for MLL/FLT3-ITD AMLs, and, potentially, other types of AMLs. Grant support: Program EXCELES, ID Project No. LX22NPO5102; GA19-09086S; MH CZ-DRO (UHKT, 00023736). Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

RNA Deadenylation Subunit CNOT3 Promotes Myeloid Leukemia By Driving Translation of Oncogenic Targets

While somatic alterations in genetic and epigenetic mechanisms in tumorigenesis have been studied extensively, how processes that affect post-transcriptional and translational regulation impact leukemia development is much less well developed. To identify novel regulators of post-transcriptional and translation control in AML, we surveyed several genome-wide CRISPR screens and observed that a number of subunits in the RNA deadenylation CNOT complex were highly ranked among genes essential for survival of human and mouse leukemia cells. The CCR4-NOT (CNOT) complex is one of two major multi-subunit polyA deadenylation complexes which mediate the shortening of the poly(A) tails. The CNOT complex was found to be recruited to m6A transcripts to mediate mRNA degradation. An involvement of the CNOT complex with RNA methylation, mRNA degradation and translation suggest a role of CNOT in bridging these processes to mediate complex gene expression regulation in pathogenesis of AML. Among the subunits of CNOT complex, we found that the highest ranked CNOT3 adaptor subunit demonstrated elevated expression in human AML cell lines (7/9 AML cell lines) and in primary AML patients (8/11 primary samples) assayed by immunoblots. Intracellular staining analysis further demonstrated that CNOT3 is highly expressed in leukemia stem cells-enriched CD34+ compartments in 13 out of 17 tested primary AML samples. Notably, high CNOT3 mRNA was associated with a poor prognosis in 3 independent AML patient cohorts i.e. TCGA-AML, Beat-AML and AMLCG 1999. Depletion of CNOT3 by shRNA-mediate knockdown and CRISPR/Cas9-mediated knockout in a panel of genetically diverse human leukemia cells (MOLM13, MV4-11, HL-60, NB4 and OCI-AML3) resulted in strong inhibition of cell growth, increased myeloid differentiation and apoptosis and significantly delayed leukemia development in vivo (median survival of 42 days, control versus 93.5 days, sh#1 and "not reached", sh#2). CNOT3 ablation also reduced survival and colony forming ability of primary AML patient cells. On the other hand, loss of CNOT3 function in normal cord-blood derived CD34+ (CB-CD34+) robustly induced myeloid differentiation without causing cell death, suggesting a potential therapeutic window to target CNOT3 in leukemia cells. In addition, overexpression of CNOT3 promoted cell growth in both CB-CD34+ and leukemia cells. Taken together, the data strongly indicates that CNOT3 promotes leukemogenesis. To uncover the mechanisms underpinning CNOT3 function in leukemia cells, we employed a CRISPR/Cas9 saturation domain mutagenesis screen by tiling the entire CNOT3 gene with sgRNAs. We identified the NOT box in the C-term domain of CNOT3 to be most critical for AML cell viability. In addition, truncation of the NOT box in CNOT3 abrogated its ability to drive proliferation in leukemia cells. This suggests a functional dependence of leukemia cells on CNOT3 interaction with the deadenylation complex via this domain. Transcriptomic and proteomic profiling of leukemia cells depleted of CNOT3 revealed an increase in p53 pathways, IFN-g immune response and downregulation of genes in G2/M checkpoint and MYC- target genes. We further observed a marked decrease in c-MYC protein upon ablation of CNOT3. We also noted the presence of c-MYC mRNA with longer poly(A) tails in CNOT3 depleted cells. However, there is no significant alternation in total abundancy and half-life of c-MYC mRNA, indicating a post-transcriptional regulation of c-MYC protein expression. In fact, depletion of CNOT3 significantly reduced the abundancy of c-MYC mRNA in the polysome-enriched and actively translated mRNAs, indicating that CNOT3 regulates translation of c-MYC. Immunoprecipitation of CNOT3 and mass spectrometry analysis of co-precipitated proteins captured proteins involved in protein synthesis including translation elongation factors and ribosomal proteins, suggesting a functional interaction of CNOT3 with the translation machinery. Overall, we uncovered a critical role of the CCR4-NOT deadenylation complex in myeloid leukemia and nominated CNOT3 as a potential target for AML therapy.

Inhibition of ERK1/2 Reverses Venetoclax-Induced Autophagy to Overcome Resistance in Acute Myeloid Leukemia

Background: Activation of the RAS/MAPK pathway confers inherent/acquired resistance to the Bcl-2 inhibitor, venetoclax, in AML (Zhang et al., Signal Transduct Target Ther. 2022). Pro-survival Bcl-2 inhibits apoptosis as well as autophagy. Phosphorylation of Bcl-2 by ERK1/2 leads to dissociation of the autophagic protein Beclin 1 from Bcl-2 and promotes autophagy (Pattingre et al., Cell. 2005). Thus, while Bcl-2 inhibition can induce apoptosis, its inhibition leads to induction of autophagy (Pattingre et al., Cell. 2005) that can protect tumor cells from organelle damage and nutrient deprivation. Objective: We hypothesized that terminal MAPK pathway inhibition using an ERK1/2 inhibitor would reverse venetoclax-induced autophagy and synergistically lead to cell death/apoptosis. Methods: We inhibited ERK1/2 using Compound 27 (Heightman et al., J Med Chem. 2018), a close analog of ASTX029, which is a dual-mechanism ERK1/2 inhibitor currently under clinical investigation in solid tumors (NCT03520075). We studied the potential synergy of ERK1/2 and Bcl-2 inhibition in decreasing cell proliferation and inducing apoptosis and in overcoming venetoclax resistance in AML preclinical models and primary patient samples (n=8). We performed simultaneous analysis of cell proliferation, stress response and DNA damage using single cell proteomics analysis to understand the mechanism of synergy. Autophagy was measured by lipidated LC3 quantification (flow cytometry and immunoblotting). Colony formation assay using NRAS-mutant PDX was performed to analyze the effect on progenitor cells. For insights into the mechanisms of synergy, we conducted large-scale proteomics analyses by single cell mass cytometry (CyTOF) and simultaneously measured cell proliferation, stress response, DNA damage and Bcl-2 family proteins. Results: Inhibition of ERK1/2 sensitized OCI-AML3 (NRAS-mutant) cells (Combination Index [CI]: 0.008) and venetoclax-resistant OCI-AML2 cells (CI: 0.05) to venetoclax and shifted the cytostatic effect of the single agents to a cytotoxic effect. Combination treatment induced apoptosis in leukemia initiating progenitor cells (LICs, CD34+CD38-) in primary AML samples with 54% apoptosis in combination versus 10% with venetoclax alone (CI = 0.03-0.23), while sparing normal CD34+CD38- cells. Mechanistically, basal ERK1/2 phosphorylation and autophagy (LC3 lipidation), were higher in venetoclax-resistant OCI-AML2 cells versus parental cells (p=0.0008) and both were inhibited with ERK1/2 inhibition alone and in combination with venetoclax. Simultaneous analysis of cell proliferation, stress response and DNA damage showed downregulation of proliferation (Ki-67) and an increase in ATF4 in response to ERK1/2 inhibition. The combination of Compound 27 and venetoclax inhibited autophagy (lipidated LC3) and increased apoptosis (increased cleaved PARP and cleaved caspase 3, Fig 1a). Venetoclax-induced autophagy was also confirmed in OCI-AML3 cells at 24 h post treatment (p=0.03). ERK1/2 inhibition reversed venetoclax-induced autophagy in OCI-AML3 (p=0.0003) and OCI-AML2-venetoclax-resistant cells (p=0.0007). CyTOF analysis showed decreased p-AMPK in response to ERK1/2 inhibition in OCI-AML3 cells. As AMPK/FIS1-mediated mitochondrial autophagy is required for self-renewal of AML stem cells (Pie et al., Cell Stem Cell. 2018), we confirmed decreased clonogenic potential of AML progenitor cells by reduced colony formation in response to ERK1/2 inhibition using an NRAS-mutant AML PDX model (p=0.0002). Single cell proteomics (CyTOF) showed decreased p-AMPK in response to single agent ERK1/2 inhibition or the combination with venetoclax in LIC populations (CD34+CD38-) in the NRAS-mutant AML PDX model. In addition to p-AMPK the expression of cMyc and CD44 was also reduced in the combination treatment, indicating reduced stemness (Fig 1b). Conclusion: Mechanistically, our results indicate that pharmacological inhibition of ERK1/2 impairs autophagy and sensitizes venetoclax-resistant AML cells to apoptosis, thus overcoming primary and secondary resistance to venetoclax. In addition, combination treatment induced apoptosis of LICs and reduced clonogenic potential and markers of stem cell function. These data provide a strong rationale for the combination of ERK1/2 and Bcl-2 inhibitors in treatment of AML and warrant further investigation in the clinic. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Sting Agonist GSK3745417 Induces Apoptosis, Antiproliferation, and Cell Death in a Panel of Human AML Cell Lines and Patient Samples

GSK3745417 is currently in phase 1 development targeting solid tumor indications. Recent literature has suggested a role for STING (Stimulator of Interferon Genes) activation induced cytotoxicity in AML cells (Baba et al, Cell Death Dis, 2021; 12(4):322.), indicating potential for therapeutic benefit in AML/MDS. To determine the implications of STING activation on tumor cells directly, GSK3745417 treatment was assessed in a screen against a panel of 93 tumor cell lines from various tumor origin. GSK3745417 treatment resulted in cytokine production across cell lines but cell growth inhibition in only 3 of the cell lines, including the only 2 AML cell lines in the panel. GSK3745417 was subsequently tested in an expanded panel of 13 AML cell lines. The data confirmed the cell growth inhibitory effect of GSK3745417 in 11/13 AML cell lines. Overall, growth inhibition was more potent than IFNβ induction, indicating a distinct growth inhibition/cytotoxicity mechanism in AML cells upon STING activation in addition to the potential to induce anti-AML immune response through type 1 IFN induction (Curran et al, Cell Rep, 2016;15(11):2357-66.). Further study demonstrated caspase induction in 7 out of 11 responding cell lines upon GSK3745417 treatment which was blocked by apoptosis inhibitor QVD. This supports the hypothesis that GSK3745417 induced cytotoxicity in AML cells is driven through an apoptosis mechanism. GSK3745417 also blocked colony formation in primary AML samples from 5 donors. GSK3745417 is able to induce growth inhibition with transient compound incubation in vitro in as little as 30 minutes to 1 hour. Taken together, this data suggests a role for GSK3745417 as a therapeutic in AML/MDS indications through dual mechanisms of direct tumor cell killing and immune activation at a clinically relevant dose. The human biological samples were sourced ethically and their research use was in accord with the terms of the informed consents under an IRB/EC approved protocol.

Safety and Efficacy of the Addition of the Protein Translation Inhibitor Omacetaxine to Induction with Cytarabine and Idarubicin in Newly Diagnosed AML

Background: Omacetaxine mepesuccinate (OM), a semi-synthetic form of homoharringtonine (HHT), mechanistically inhibits aminoacyl-tRNA binding to ribosomal acceptor "A” sites, preventing mRNA translation (Wetzler,Curr Pharm Des 2011, 17:59-64), and causing a rapid decline of short-lived proteins e.g., c-myc, cyclin D, the anti-apoptotic proteins cFLIP, survivin and Mcl-1, an important anti-apoptotic regulator in AML.While HHT added to cytarabine and an anthracycline improved CR and 3-yr EFS in de novo AML patients (pts; Jin et al, Lancet Oncology 2013, 7:599-608), meta-analysis of Chinese studies in elderly pts indicated a CR of 47.5%. Aims: Early analysis of AML-02, a phase Ib trial of escalating doses of OM (an FDA-approved drug) added to a standard 7 + 3 induction regimen was described (NCT02440568; Christian et al., Blood, 2018, 132:5218). Here we report long-term pt outcomes and studies of their correlation with in vitro OM effects on protein translation and apoptosis sensitivity. Methods: From June 2015 to June 2018, 22 pts aged 18-70 yrs with newly diagnosed AML entered AML-02, receiving escalating doses of OM administered SQ q12h d1-7 with cytarabine (100mg/m2 CIV) d1-7 and Idarubicin 12mg/m2 IV d1-3. Four OM dose levels were tested starting with cohort #1 at 0.625mg/m2 sc q12h (also 1.25, 2.0, and 3.0mg/m2 q12h cohorts). The primary endpoint, the optimally safe and active dose (OD) was determined using a Bayesian adaptive design, EFFTOX, which considers trade-offs between efficacy and toxicity to establish the OD (Shah et al, Leukemia, 2015; 29:1945). Secondary endpoints included OS, RFS and EFS. Residual disease at d14 was deemed a study failure and pts were taken off study. Hematological toxicity (HT) was defined as incomplete hematologic recovery at d49 with the BM free of leukemia. Post-induction therapy consisted of standard high dose cytarabine consolidation chemotherapy or allogeneic HSCT, based on cytogenetic risk (ELN 2010). Potential correlations of the effects of OM on protein translation rates, measured using the OP-Puro technique, and apoptosis sensitivity, measured using BH3 profiling techniques, on pretreatment AML samples and subsequent patient outcomes were also examined. Follow-up is complete to August 1st, 2022. Results: Median age was 58 yrs, 12 pts had adverse cytogenetics, 6 intermediate and 3 favorable risk (1 unknown due to fibrosis); overall 73% (16) had adverse cytogenetics or secondary AML. Earlier results (Christian et al) indicated no HTs in 21 evaluable pts, and OD was established as 2.0mg/m2. CR was 45.5%, median OS 22.7 months (OS at 4 yrs 45%), and EFS 80 days. The median OS is now 24.2 months (95% CI 18.3-39.7), median EFS is 80 days (95% CI 280-964), and OS at 6 years remains at 45%. There were no new TEAEs. Notably, of 10 pts achieving CR, 1 pt relapsed at D60, 1 was lost to follow-up and 1 died of opiate overdose (in CR). Five of the remaining 7 underwent HSCT and all 7 remain in remission. In pts achieving CR, median RFS is 62.4 months (95% CI 42.6-82.2; Fig.1), suggesting potential LSC impact. Common adverse effects included neutropenia (82%), nausea (64%), vomiting (59%), diarrhea (45%), hyperglycemia (41%), and a transient desquamating skin reaction (27%) that is dose-related and may be due to OM-mediated downregulation of cFLIP levels in sweat glands (Hwang et al, British Journal of Haematology, 2021; 195:e138-e146). In in vitro studies, 3 pretreatment AML pt BM samples were analyzed after a short-term (non-cytotoxic) OM treatment for 4 hours at a dose (75 nM) equivalent to calculated systemic OM levels achieved in cohort#3 (2mg/m2). Responding pt samples (CR;n=2) had a greater reduction in protein translation than the non-responder (NR). With exposure to OM, CR pts also had greater reductions of proteins with a short T1/2, such as anti-apoptotic protein MCL-1, with a marked increase in cytochrome C loss, indicating high mitochondrial priming and sensitivity to apoptosis (Fig.2). Conclusion: This small phase Ib study of the addition of OM to a standard 7 + 3 regimen in a diverse population with predominantly high-risk AML and frequent co-morbidities demonstrates a manageable safety profile, acceptable efficacy and resulted in a prolonged DoR. Studies of pretreatment pt samples suggest that analysis of blast protein translation inhibition and especially BH3 profiling responses to OM may predict susceptibility of LSC to Omacetaxine, and potentially patient outcomes. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Loss of FBXO11 Function Establishes a Stem Cell Program in Acute Myeloid Leukemia through Dysregulation of the Mitochondrial Protease LONP1

Despite advances in targeted therapies, survival for AML patients remains poor. Molecular classification systems attempt to stratify patients into risk groups, but 20-40% of patients do not fall under established genomic subgroups (Papaemmanuil et al., N Engl J Med 2016) We analyzed a total of 1,627 unique patient samples from the AML-PMP, TCGA, Beat AML, and TARGET datasets and identified loss-of-function mutations in ubiquitin ligase family genes in 10% of patient samples (n = 163). Among recurrently mutated genes, expression of FBXO11 was the most significantly reduced component of the SKP1-CUL1-Fbox (SCF) E3 ligase complex in AML samples compared to normal CD34+ hematopoietic stem/progenitor cells (HSPC). RNA sequencing (RNA-seq) following knockdown (KD) of FBXO11 by shRNA in cord blood derived CD34+ HSPCs resulted in enrichment of hematopoietic stem (HSC) and leukemia stem cell (LSC) signatures accompanied by a diminishment in hematopoietic progenitor cell signatures. Consistent with enrichment of a quiescent stem cell phenotype, cell cycle analysis showed that FBXO11 KD increased the G0 fraction of the CD34+ population. In liquid culture, FBXO11 KD HSPCs displayed myeloid-biased output. Fbxo11 KD in mouse HSPCs co-operated with AML1-ETO (RUNX1-RUNX1T1) fusion to generate serially transplantable AML in mice. The combination of FBXO11 KD with AML1-ETO and a KRASG12D mutant in human cord blood HSPC also promoted stem cell maintenance and myeloid malignancy in a human xenotransplant model, but AML1-ETO with KRASG12D without FBXO11 KD did not. Mass spectrometry analysis of proteins co-immunoprecipitating with FLAG-FBXO11 in myeloid cells identified the the mitochondria protease and chaperone protein LONP1. LONP1 protein expression remained unchanged with FBXO11 overexpression or CRISPR/Cas9-mediated targeting, suggesting that LONP1 is not targeted for degradation by the SCFFBXO11 complex. FBXO11 has been demonstrated to facilitate ubiquitination (Xu et al., Blood 2021) and neddylation (Abida et al., J Biol Chem 2007) of protein targets. FBXO11 expression resulted in activating K63 ubiquitination as well as neddylation of LONP1, but only ubiquitination inhibition resulted in reversal of an FBXO11-overexpression phenotype. Further, proteasome inhibitors also did not reverse the FBXO11-overexpression phenotype, consistent with FBXO11 mediating an activating ubiqutination of LONP1. FBXO11 or LONP1 KD in cord blood HSPCs promoted myeloid biased output and reduced mitochondria membrane potential, a characteristic of long-term HSCs (Mansell et al., Cell Stem Cell 2021). To validate these findings and direction of the FBXO11-LONP1 relationship, we performed RNA-seq on cord blood HSPCs following lentiviral transductions to combinatorially deplete or overexpress FBXO11 and LONP1. Unsupervised clustering of the samples demonstrated overexpression of LONP1 clustered with controls while knockdown of LONP1 reversed the effect of FBXO11 overexpression. We observed upregulation of a mitochondria respiratory defect gene set with depletion of FBXO11 or LONP1, indicating improper assembly of the electron transport chain, a reported consequence of impaired LONP1 function (Ghosh et al., Oncogene 2019). We also determined enrichment in several HSC and LSC genesets with KD of either FBXO11 or LONP1, while overexpression of FBXO11 downregulated these genesets. In contrast to normal HSCs which utilize glycolysis for energy production (Simsek et al., Cell Stem Cell 2010), LSCs from AML patients, are dependent on oxidative phosphorylation for energy metabolism but remain in a quiescent state (Jones et al., Cancer Cell 2018). Interestingly, FBXO11 depletion resulted in downregulated glycolysis genesets and upregulated quiescent stem cell signatures. Taken together, these findings suggest that FBXO11 depletion dysregulates LONP1 function to establish a mitochondrial state reminiscent of quiescent HSC. In summary, we demonstrate that the ubiquitin ligase family is frequently mutated in AML. We show that gene expression of a component of the SCF E3 ubiquitin ligase, FBXO11, is reduced in AML, and that FBXO11 depletion cooperates with other driver mutations to initate AML in mouse and human xenotransplant models. Finally, we establish a novel link between FBXO11 and LONP1, and provide evidence that loss of FBXO11 regulation of LONP1 promotes a myeloid-biased HSC/LSC-like state.

The Exportin, XPO2, Is a Novel Therapeutic Target in Pediatric, Adolescent and Young Adult AML

The active transport of proteins across the nuclear membrane is regulated by nuclear transport receptors termed importins and exportins, depending on their direction of transport. Nuclear transport receptors contribute to cancer development, through abnormal localization of protein cargos. Increased expression of the exportin, exportin-1/chromosome region maintenance 1 (XPO1/CRM1), has been associated with high risk AML and shorter survival times and XPO1 inhibitors show signs of efficacy in AML. While previous data demonstrate a functional role for XPO1 in AML, less is known about the exportin, XPO2, (alias CSE1L). Despite XPO2 RNAi depletion screens demonstrating a higher genetic dependence in leukemia versus solid tumors/non-hematologic malignancies (p = 0.0394), the functional impact of inhibiting XPO2 has never been studied in leukemia. We analyzed a public database of CRISPR/Cas9 depletion screens in 26 AML cell lines. XPO2 ranked in the top 1.4% of essential genes in AML, along with KPNB1 and XPO1 (DepMap 22Q2 Public+Score, Chronos, www.depmap.org). An RNAi depletion screen confirmed these findings, with XPO2 identified within the top 0.2% of essential genes in AML (Achilles+DRIVE+Marcotte, DEMETER2, www.depmap.org). Gene dependency scores indicated a greater reliance on XPO2, compared to XPO1 (p <0.0001). Increased expression of XPO2 mRNA was associated with decreased event free survival (p = 0.0014) and overall survival (p = 0.00091) in pediatric and adolescent/young adult (AYA) AML patients from the TARGET dataset. Analyzing the BeatAML dataset, increased XPO2 mRNA expression was also associated with decreased overall survival in AML patients <40 years, but not in patients >40 years (p <0.0001). We analyzed protein expression databases and identified XPO2 as the highest expressed member of the exportin family in AML cells lines and AML primary patient samples. By immunoblot analysis, increased expression of XPO2 was detected in 7/7 primary AML samples and 8 AML cell lines (HL60, MV4-11, MOML13, NB4, OCI-AML2, OCI-M2, TEX and U937), compared to normal hematopoietic cells. XPO2 mRNA was also increased in 5/5 primary AML samples and 8/8 AML cell lines, compared to normal hematopoietic cells, as measured by qRT-PCR. To confirm the functional importance of XPO2 in AML, we knocked down XPO2 with shRNA in AML cell lines. Genetic silencing of XPO2 decreased growth and viability of OCI-AML2, TEX and NB4 cells. Gambogic acid (GA), a natural product from Garcinia hanburyi, has been identified as a covalent inhibitor of XPO2-mediated nuclear transportation. In OCI-AML2, TEX and NB4 cell lines, gambogic acid decreased growth and viability of AML cell lines at nanomolar concentrations (TEX cells IC50 = 369nM, OCI-AML2 cells IC50 = 433nM, NB4 cells IC50 = 161nM). To uncover the mechanism of how XPO2 mediates cell death in AML we interrogated a publicly available BioID database (PXD007976) and performed gene set enrichment analysis on XPO2 targets using gprofiler. Processes related to ribosome biogenesis were significantly enriched in XPO2 cargo, whereas XPO1 cargo were enriched for processes related to snRNA transcription and transport (p <0.05). In summary, we identified XPO2 as an essential gene for AML. XPO2 is preferentially increased in AML cell lines and primary patient samples compared to normal hematopoietic cells. Increased XPO2 expression is associated with worse clinical outcomes in pediatric and AYA patients with AML. XPO2 may function to enhance ribosomal biogenesis, demonstrating a critical role in leukemia initiation and progression. Thus, this work demonstrates a new dependency of AML on XPO2, as well as highlights a new therapeutic target for this disease.

ERK1/2 Inhibition Overcomes Resistance to Venetoclax in AML By Altering Drp1-Dependent Mitochondrial Fission and Metabolism

Background: Mutational (RAS, PTPN11 etc.) or non-mutational activation of RAS/RAF/ERK pathway is associated with primary or secondary resistance to venetoclax in AML therapy (Zhang et al., Sig Transduct Target Ther. 2022). While upregulation of alternate antiapoptotic proteins e.g. Mcl-1 has been implicated in resistance to venetoclax (Zhang et al., Sig Transduct Target Ther. 2022) role of mitochondrial dynamics (fission/fusion) in venetoclax resistance has not been well explored. Oncogenic RAS activates downstream ERK1/2 phosphorylation and mitochondrial fission through ERK1/2-mediated phosphorylation of the dynamin-like mitochondrial fission GTPase Drp1 (Nagdas et al., Cell Rep. 2019). Drp1 deletion inhibits tumorigenesis of KRAS driven pancreatic cancer, but the importance of RAS driven changes in mitochondrial dynamics in AML is poorly defined. ERK1/2 inhibition may therefore provide insights into the dependence of AML cells on altered mitochondrial dynamics and metabolism driven by oncogenic RAS. Objectives: We assessed the synergistic anti-leukemia efficacy of concomitant Bcl2 and ERK1/2 inhibition in preclinical models of venetoclax resistance and in primary patient samples (n=8). Further, we investigated the role of ERK1/2/Drp1-dependent mitochondrial fission in mediating resistance to venetoclax through altered metabolism. Results: ERK1/2 inhibition using Compound 27 (Heightman et al., J Med Chem. 2018) a close analog of ASTX029, which is currently under clinical investigation in solid tumors (NCT03520075), was highly synergistic with venetoclax at inducing apoptosis in AML cells. NRAS-mutant OCI-AML3 and THP1 cells, that are intrinsically resistant to venetoclax, were sensitized to venetoclax by ERK1/2 inhibition with a combination index (CI) of 0.008 (cell death ~90% for combination versus 20% with venetoclax alone). Synergy was also confirmed in OCI-AML2 cells with acquired resistance to venetoclax, resulting in 75% apoptosis versus 22% by venetoclax alone (CI=0.6). The combination was also effective in eliminating bulk and CD34+CD38- leukemia initiating cells (LICs) in primary AML samples (CI:0.03-0.23). Colony formation assay and single cell level CyTOF analysis using an NRAS-mutant PDX showed markedly impaired clonogenic potential (p=0.0002) and decreased expression of cMyc and CD44 in LICs in response to single agent ERK1/2 inhibition and combination treatment, indicating a potential impact on stemness. Mitochondrial reactive oxygen species (ROS) mitigation, Drp1-mediated mitochondrial fission and glycolysis are critical to maintain stemness in AML (Schimmer et al., Cell Stem Cell. 2018). As a result of ERK1/2 inhibition, pDrp1 (Ser616) levels decreased, and mitochondrial length (p<0.001) and mitochondrial ROS levels (p<0.01) were increased, suggesting impaired fission. In OCI-AML3 cells, combination treatment caused downregulation of Drp1 mRNA and decreased phosphorylation of Drp1 at Ser616. Rescue with overexpression of active phospho-mimetic form of Drp1 i.e. Drp1-Ser616Glu-Ser637Ala in OCI-AML3 cells resulted in decreased apoptosis and reversed the synergy between ERK1/2 and Bcl-2 inhibition when compared to wild type and phospho-null (Ser616Ala) Drp1 overexpression (p<0.001, Fig 1a). Of note, overexpression of Drp1-Ser616Glu-Ser637Ala phospho-mimetic led to decreased mitochondrial length (Fig 1b), suggesting enhanced fission with a distinct metabolic phenotype including reduced basal respiration, decreased maximal oxygen consumption rate (p<0.05) and ATP production (p<0.05), as well as reduced ROS production (p<0.01). Moreover, combination treatment failed to significantly induce ROS production in cells expressing the phospho-mimetic Drp1 as opposed to those expressing phospho-null or wildtype Drp1. Collectively, these results indicate that inhibition of ERK1/2/Drp1dependent mitochondrial fission and metabolic alterations may be a potential mechanism of synergistic tumor cell killing for the combination treatment. Conclusion: ERK1/2 inhibition alters Drp1 dependent mitochondrial dynamics and metabolism, resulting in mitochondrial defects and overcoming resistance to venetoclax by causing apoptosis in venetoclax resistant AML cells. These data provide a strong rationale for the combination of ERK1/2 and Bcl-2 inhibitors in treatment of AML and warrant further investigation in the clinic. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

139. Feasibility of comprehensive whole genome profiling in hematological malignancies

Whole genome sequencing (WGS) has been recently proposed as a replacement for routine karyotype and FISH analyses of myeloid and lymphoid neoplasms. WGS allows for the unbiased and comprehensive detection of multiple types of genomic aberrations, from single nucleotide to structural variants, which could streamline current genomic testing algorithms and clinical laboratory workflows for hematologic malignancies. In this study, we explored the feasibility of whole genome profiling of myeloid and lymphoid neoplasms by sequencing eleven AML, MPN, MDS, B-ALL, and T-ALL genomes with an average depth of sequencing coverage of ∼40X, and comparing genome analyses data to their available cytogenetic and molecular panel results. 81% of clinically relevant SNVs were detected by WGS, with false negatives being mostly variants at low frequencies (VAF <10%). Nine new variants were detected by WGS in an AML, B-ALL and T-ALL samples, eight of which were classified as VUS, and one (FLT3 p.Tyr572Cys) in B-ALL as likely pathogenic. 95% of clinically relevant copy-number variants (CNVs) were detected by WGS, with false negatives being mostly small (<150Kb) or mosaic CNVs present in <15% of the sample. The identification of the clinically relevant translocations in these samples (such as ETV6/RUNX1 and CRLF2 rearrangements) using WGS data is currently being investigated. This feasibility study demonstrated the potential of using WGS as the replacement of molecular panels and chromosome microarray in hematologic malignancies, particularly at the time of initial diagnosis.

S122: ROCK INHIBITORS TARGET SRSF2 LEUKEMIA BY DISRUPTING CELL MITOSIS AND NUCLEAR MORPHOLOGY

Background: Spliceosome machinery mutations are common early mutations in myeloid malignancies, however effective targeted therapies against them are still lacking in clinical settings. Aims: Exploring safe and efficient methods to target hematopoietic cells carrying SRSF2 mutations might be a powerful tool not just for leukemia prevention and treatment, but also to understand the mechanisms behind SRSF2 mutations. Methods: We generated five SRSF2 Mut (P95H) isogenic cell lines with CRISPR/CAS9 system and performed high throughput drug screen with 3988 compounds in a single dose. After we narrowed down our targets, dose response assay was performed on four different ROCK inhibitors (ROCKi). The leading ROCKi compound was validated in vivo with SRSF2 Mut AML xenograft models. Next, we aimed at understanding why ROCKi target SRSF2 Mut cells and used proteomics, gene expression and imaging of the cytoskeletal system to study the effect of ROCKi on Mut and WT cells. Results: In the current study, we used an in vitro high-throughput drug screen among four different isogenic cell lines and identified ROCK inhibitors (ROCKi) as selective inhibitors of SRSF2 Mut in MOLM14 and AML2 cells. To study the efficacy of RKI-1447 on human samples, we conducted six AML patient-derived xenograft (PDX) experiments, with SRSF2 Mut samples. In four out of the six primary AML samples, RKI-1447 significantly reduced engraftment compared to the untreated group and inhibited the engraftment of both leukemic blasts and pre leukemic-HSPCs (preL-HSPCS). RKI-1447 was not toxic to mice nor human cells. ROCKi induced mitotic catastrophe (G2M arrest and multipolar spindles) through their apparent effects on microtubules and nuclear organization, and leading to cell death. Confocal imaging and transmission electron microscopy (TEM) data revealed that SRSF2 mutations induce deep nuclear indentation and segmentation, which is reminiscent of the nuclear shape of Pelger–Huët anomaly (PHA). The nuclear volume and area were significantly higher in SRSF2 Mut compared with WT MOLM14 cells regardless of ROCKi. To investigate why SRSF2 Mut cause the structural changes, we looked into the cytoplasmic intrusions that segment the nuclei, and the structures that connect the lobes of the nuclei by TEM. This examination revealed enrichment of microtubule bundles inside the nuclear indentations. More importantly, these microtubules were located at the tip of the cytoplasmic intrusions suggesting that they take an active role in the nuclear segmentation process. After exposure to RKI-1447, the cytoskeletal and nuclear morphological abnormalities of SRSF2 Mut are augmented to a level which are incompatible with cell survival. Image:Summary/Conclusion: We believe it is the first report describing in high resolution the changes in the nucleus after SRSF2 mutations are introduced. The mechanisms we identified are most probably relevant to other SMMs and to the dysplastic phenotype observed in MDS and AML. Accordingly, our findings have wide implications as ROCKi might be even more useful than describe here. Altogether, we provide data from many directions that ROCKi should be tested in clinical trials.

P456: A LONG READ SEQUENCING AND CRISPR-CAS9 BASED APPROACH FOR RAPID COPY-NUMBER ALTERATION AND STRUCTURAL VARIATION DETECTION IN HEMATOLOGIC MALIGNANCIES

Background: Current standard karyotyping methods are labor-intensive and have severe limitations in terms of resolution and duration to results. For hematologic malignancies, but not restricted to, this represents a relevant obstacle in translating molecular findings into time critical treatment decisions. Aims: To develop a sequencing based approach for rapid and scalable cytogenetics that can be applied to a wide spectrum of hematologic malignancies to detect clinically relevant molecular markers within 48hrs Methods: For copy number alteration detection, whole genome sequencing was performed on a GridION sequencer (Oxford Nanopore Technologies, ONT, using the SQK-LSK109 kit) in a cohort of 53 hematologic neoplasms, including acute myeloid leukemia (AML, n=18), chronic lymphocytic leukemia (CLL, n=8), multiple myeloma (MM, n=17) and pediatric acute lymphocytic leukemia (ALL, n=10) samples. For the identification of balanced alterations, transcriptome sequencing data were generated using ONT (SQK-DCS109 kit), and we applied an in-house developed analysis pipeline based on minimap2 alignment followed by Blast with an ensuing filtering algorithm based on orientation, inter-read distance and length filtering. For targeted sequencing of structural aberrations, we developed a CRISPR-Cas9 based tiling approach using sgRNA pools (Integrated DNA Technologies, IDT). This approach allows to cover AML relevant targets such as t(8;21), the KMT2A (MLL) breakpoint area, and the FLT3-ITD region as well as lymphoma relevant regions such as the immunoglobulin heavy chain locus (library preparation was performed using SQK-CS9109 kit). Results: For copy number profiling a median whole-genome coverage of 2.6 was reached (range 0.27-7 fold). ONT sequencing and conventional karyotyping approaches showed a high concordance with Pearson correlation coefficients >0.95 for copy number alteration comparisons between conventional cytogenetics and ONT sequencing results for all investigated disease entities. Regarding the detection of structural aberrations transcriptome sequencing and fusion gene analysis using the above described analysis and filtering pipeline we could e.g. reliably detect the t(9;22) translocation from the K562 cell line and the t(8;21) translocation from Kasumi-1. Additionally, using this method we were able to correctly identify the primary AML sample harboring a t(8;21) translocation that was among the n=12 AML patient samples. In addition to an RNA-based work-flow, we established a CRISPR-Cas9 based DNA enrichment approach for the detection of recurrent structural aberrations from specified genomic DNA loci. The enrichment of genomic regions of interest using sgRNA libraries led e.g. to a median coverage of 44 reads (range 5-136-fold) of the t(8;21) region-of-interest in primary AML samples, which allow the detection of the exact coordinates of the breakpoints. Summary/Conclusion: Long read based sequencing based copy number alteration and structural variation detection based on combination of different long read sequencing approaches like low-coverage whole genome sequencing, transcriptome sequencing and CRISPR-Cas9 based sequencing of genomic loci of interest represents a highly promising tool for high resolution and high speed cytogenetics that has the potential to overcome many limitations of conventional cytogenetics.

PB1790: ABCB1 AS A POTENTIAL BENEFICIAL TARGET OF MIDOSTAURIN IN ACUTE MYELOID LEUKEMIA

Background: In AML, therapeutic outcome of anthracycline-based induction therapy often can be compromised by a resistant phenotype associated with overexpression of ABCB1 transporters. Novel anti-AML agent, midostaurin, was designed to aim at multiple targets including FLT3 mutation. Besides, it was also identified as ABCB1 inhibitor. Aims: To evaluate ABCB1 efflux activity and expression in primary AML samples in relation to multiple clinical parameters and investigate ABCB1 regulation by microRNA. Methods: ABCB1 gene expression was absolutely quantified in 28 primary AML samples collected prior to any treatment. Efflux activity was examined using combination of mitoxantrone (ABCB1 substrate) and midostaurin (ABCB1/FLT3 inhibitor). Results were compared in molecular classes defined by CD34 marker, FLT3-ITD and NPM1 mutations, ELN risk classification and (un)achieved complete remission (CR). Enhancement of daunorubicin proapoptotic effect mediated by midostaurin was determined in HL-60 wt and HL-60 overexpressing ABCB1. ABCB1 regulation by microRNAs (miR-9, miR-27a, miR-331) was evaluated by ddPCR and correlated with ABCB1 efflux activity. Results: High ABCB1 expression was associated with unachieved CR, adverse ELN risk and CD34 positivity. ABCB1 efflux activity was observed in a functional study when mitoxantrone accumulation increased as a direct consequence of midostaurin in CD34+ AML and patients with unachieved CR. ABCB1 efflux activity and expression were found to be linked to miR-9 downregulation Summary/Conclusion: We propose therapeutic value of midostaurin for drug-resistant patients beyond the FLT3 inhibition based on ABCB1 expression and CD34 positivity. We also propose miR-9 as a predictive ABCB1-related biomarker that could be helpful in identifying ABCB1-resistant AML phenotype.

Abstract 2864: A CLEC12A immunocytokine with target cell-restricted IL-15 activity shows a favorable toxicity profile and high potency in AML

Abstract IL-15, often referred to as the “anti-cancer cytokine”, potently stimulates proliferation and activation of NK and T cells, but unlike its close relative IL-2, does neither promote activation-induced death of lymphocytes nor activity of immunoinhibitory Tregs. So far, short half-life, poor accumulation at the tumor site and severe toxicity upon systemic application limit IL-15 efficacy in patients. Fusion of IL-15 to antibodies directed to tumor antigens (classical immunocytokines, ICs) improves on accumulation at the tumor site and pharmacokinetics. However, since the activity of the cytokine moiety within classical ICs does not depend on antigen binding, the application of clinically effective doses is still prevented by toxicity due to unspecific immune activation. To overcome this problem, we took advantage of the unique mechanism of action of IL-15 which stimulates IL-15Rβ/γ on NK and T cells as a membrane-bound complex with IL-15Rα on monocytes and DCs (trans-presentation). We used an Fc-optimized antibody directed to CLEC12A, a surface antigen abundantly expressed on AML cells and leukemic stem cells but not on healthy stem cells. This antibody termed 33C2-SDIE was fused to an IL-15E46K mutant with abolished binding to IL-15Rα, allowing to substitute physiological trans-presentation of IL-15 by binding of the construct to its tumor-expressed target. Antigen-specific binding of the resulting modified immunocytokine (MIC12) was confirmed using multiple CLEC12A-expressing cell lines and primary AML samples from patients. Functional analysis of activation, cytokine release, and target cell lysis demonstrated that MIC proteins, in contrast to classical IC, stimulate cytotoxic lymphocytes in a highly target cell-restricted manner, allowing for the desired reduction of unspecific immune activation. At the same time, significantly superior NK cell reactivity against AML cells as compared to even the Fc-optimized antibody was observed. Both, IL-15 signaling and engagement of Fc-receptors by the optimized Fc-domain were found to be essential for optimal activity of our MIC constructs. Importantly, only treatment with MIC12 was capable to induce NK cell proliferation, which is required to overcome unfavorable target to effector ratios that prevail in overt cancer disease. In summary, our novel CLEC12A-targeting immunocytokine allows for tumor-restricted stimulation of cytotoxic lymphocytes and reduced toxicity while displaying superior anti-leukemic activity and constitutes a promising compound for the treatment of AML. Citation Format: Boris Klimovich, Leonard Anton, Yangmi Lim, Jonghwa Won, Anna Chashchina, Martin Pflügler, Latifa Zekri, Gundram Jung, Helmut R. Salih. A CLEC12A immunocytokine with target cell-restricted IL-15 activity shows a favorable toxicity profile and high potency in AML [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2864.

A multiparametric niche-like drug screening platform in acute myeloid leukemia

Functional precision medicine in AML often relies on short-term in vitro drug sensitivity screening (DSS) of primary patient cells in standard culture conditions. We designed a niche-like DSS assay combining physiologic hypoxia (O2 3%) and mesenchymal stromal cell (MSC) co-culture with multiparameter flow cytometry to enumerate lymphocytes and differentiating (CD11/CD14/CD15+) or leukemic stem cell (LSC)-enriched (GPR56+) cells within the leukemic bulk. After functional validation of GPR56 expression as a surrogate for LSC enrichment, the assay identified three patterns of response, including cytotoxicity on blasts sparing LSCs, induction of differentiation, and selective impairment of LSCs. We refined our niche-like culture by including plasma-like amino-acid and cytokine concentrations identified by targeted metabolomics and proteomics of primary AML bone marrow plasma samples. Systematic interrogation revealed distinct contributions of each niche-like component to leukemic outgrowth and drug response. Short-term niche-like culture preserved clonal architecture and transcriptional states of primary leukemic cells. In a cohort of 45 AML samples enriched for NPM1c AML, the niche-like multiparametric assay could predict morphologically (p = 0.02) and molecular (NPM1c MRD, p = 0.04) response to anthracycline-cytarabine induction chemotherapy. In this cohort, a 23-drug screen nominated ruxolitinib as a sensitizer to anthracycline-cytarabine. This finding was validated in an NPM1c PDX model.

Regulation of proton partitioning in kinase-activating acute myeloid leukemia and its therapeutic implication

Gain-of-function kinase mutations are common in AML and usually portend an inferior prognosis. We reported a novel mechanism whereby kinase mutants induced intracellular alkalization characteristic in oncogenesis. Thirteen kinases were found to activate sodium/hydrogen exchanger (NHE1) in normal hematopoietic progenitors, of which FLT3-ITD, KRASG12D, and BTK phosphorylated NHE1 maintained alkaline intracellular pH (pHi) and supported survival of AML cells. Primary AML samples with kinase mutations also showed increased NHE1 phosphorylation and evidence of NHE1 addiction. Amiloride enhanced anti-leukemic effects and intracellular distribution of kinase inhibitors and chemotherapy. Co-inhibition of NHE1 and kinase synergistically acidified pHi in leukemia and inhibited its growth in vivo. Plasma from patients taking amiloride for diuresis reduced pHi of leukemia and enhanced cytotoxic effects of kinase inhibitors and chemotherapy in vitro. NHE1-mediated intracellular alkalization played a key pathogenetic role in transmitting the proliferative signal from mutated-kinase and could be exploited for therapeutic intervention in AML.

Cystine uptake inhibition potentiates front-line therapies in acute myeloid leukemia

By querying metabolic pathways associated with leukemic stemness and survival in multiple AML datasets, we nominated SLC7A11 encoding the xCT cystine importer as a putative AML dependency. Genetic and chemical inhibition of SLC7A11 impaired the viability and clonogenic capacity of AML cell lines in a cysteine-dependent manner. Sulfasalazine, a broadly available drug with xCT inhibitory activity, had anti-leukemic activity against primary AML samples in ex vivo cultures. Multiple metabolic pathways were impacted upon xCT inhibition, resulting in depletion of glutathione pools in leukemic cells and oxidative stress-dependent cell death, only in part through ferroptosis. Higher expression of cysteine metabolism genes and greater cystine dependency was noted in NPM1-mutated AMLs. Among eight anti-leukemic drugs, the anthracycline daunorubicin was identified as the top synergistic agent in combination with sulfasalazine in vitro. Addition of sulfasalazine at a clinically relevant concentration significantly augmented the anti-leukemic activity of a daunorubicin-cytarabine combination in a panel of 45 primary samples enriched in NPM1-mutated AML. These results were confirmed in vivo in a patient-derived xenograft model. Collectively, our results nominate cystine import as a druggable target in AML and raise the possibility to repurpose sulfasalazine for the treatment of AML, notably in combination with chemotherapy.

Next-Generation DNA Sequencing-Based Gene Panel for Diagnosis and Genetic Risk Stratification in Onco-Hematology.

Simple SummaryThe present work comes up after detecting stakeholders’ need to test for manifold molecular biomarkers in each sample from an individual diagnosed with myeloid neoplasm or acute leukemia. The development of gene panels based on NGS technology is considered a potentially effective testing alternative with less human effort, compared to that required by other conventional techniques (PCR, FISH, conventional karyotype, etc.). The validation of this panel aims to propose a new solution for hospitals to face the challenges posed by the molecular study of this group of onco-hematological diseases.A suitable diagnostic classification of myeloid neoplasms and acute leukemias requires testing for a large number of molecular biomarkers. Next-generation sequencing is a technology able to integrate identification of the vast majority of them in a single test. This manuscript includes the design, analytical validation and clinical feasibility evaluation of a molecular diagnostic kit for onco-hematological diseases. It is based on sequencing of the coding regions of 76 genes (seeking single-nucleotide variants, small insertions or deletions and CNVs), as well as the search for fusions in 27 target genes. The kit has also been designed to detect large CNVs throughout the genome by including specific probes and employing a custom bioinformatics approach. The analytical and clinical feasibility validation of the Haematology OncoKitDx panel has been carried out from the sequencing of 170 patient samples from 6 hospitals (in addition to the use of commercial reference samples). The analytical validation showed sensitivity and specificity close to 100% for all the parameters evaluated, with a detection limit of 2% for SNVs and SVs, and 20% for CNVs. Clinically relevant mutations were detected in 94% of all patients. An analysis of the correlation between the genetic risk classification of AML (according to ELN 2017) established by the hospitals and that obtained by the Haematology OncoKitDx panel showed an almost perfect correlation (K = 0.94). Among the AML samples with a molecular diagnosis, established by the centers according to the WHO, the Haematology OncoKitDx analysis showed the same result in 97% of them. The panel was able to adequately differentiate between MPN subtypes and also detected alterations that modified the diagnosis (FIP1L1-PDGFRA). Likewise, the cytogenetic risk derived from the CNV plot generated by the NGS panel correlated substantially with the results of the conventional karyotype (K = 0.71) among MDS samples. In addition, the panel detected the main biomarkers of prognostic value among patients with ALL. This validated solution enables a reliable analysis of a large number of molecular biomarkers from a DNA sample in a single assay.

Pseudo-mutant P53 is a unique phenotype of DNMT3A-mutated pre-leukemia.

Pre-leukemic clones carrying DNMT3A mutations have a selective advantage and an inherent chemoresistance, however the basis for this phenotype has not been fully elucidated. Mutations affecting the gene TP53 occur in pre-leukemic hematopoietic stem/progenitor cells (preL-HSPC) and lead to chemoresistance. Many of these mutations cause a conformational change and some of them were shown to enhance self-renewal capacity of preL-HSPC. Intriguingly, a misfolded P53 was described in AML blasts that do not harbor mutations in TP53, emphasizing the dynamic equilibrium between wild-type (WT) and "pseudo-mutant" conformations of P53. By combining single cell analyses and P53 conformation-specific monoclonal antibodies we studied preL-HSPC from primary human DNMT3A-mutated AML samples. We found that while leukemic blasts express mainly the WT conformation, in preL-HSPC the pseudo-mutant conformation is the dominant. HSPC from non-leukemic samples expressed both conformations to a similar extent. In a mouse model we found a small subset of HSPC with a dominant pseudo-mutant P53. This subpopulation was significantly larger among DNMT3AR882H-mutated HSPC, suggesting that while a pre-leukemic mutation can predispose for P53 misfolding, additional factors are involved as well. Treatment with a short peptide that can shift the dynamic equilibrium favoring the WT conformation of P53, specifically eliminated preL-HSPC that had dysfunctional canonical P53 pathway activity as reflected by single cell RNA sequencing. Our observations shed light upon a possible targetable P53 dysfunction in human preL-HSPC carrying DNMT3A mutations. This opens new avenues for leukemia prevention.

Hypermethylation of RAD9A intron 2 in childhood cancer patients, leukemia and tumor cell lines suggest a role for oncogenic transformation.

Most childhood cancers occur sporadically and cannot be explained by an inherited mutation or an unhealthy lifestyle. However, risk factors might trigger the oncogenic transformation of cells. Among other regulatory signals, hypermethylation of RAD9A intron 2 is responsible for the increased expression of RAD9A protein, which may play a role in oncogenic transformation. Here, we analyzed the RAD9A intron 2 methylation in primary fibroblasts of 20 patients with primary cancer in childhood and second primary cancer (2N) later in life, 20 matched patients with only one primary cancer in childhood (1N) and 20 matched cancer-free controls (0N), using bisulfite pyrosequencing and deep bisulfite sequencing (DBS). Four 1N patients and one 2N patient displayed elevated mean methylation levels (≥ 10 %) of RAD9A. DBS revealed ≥ 2 % hypermethylated alleles of RAD9A, indicative for constitutive mosaic epimutations. Bone marrow samples of NHL and AML tumor patients (n=74), EBV (Epstein Barr Virus) lymphoblasts (n=6), tumor cell lines (n=5) and FaDu subclones (n=13) were analyzed to substantiate our findings. We find a broad spectrum of tumor entities with an aberrant methylation of RAD9A. We detected a significant difference in mean methylation of RAD9A for NHL versus AML patients (p ≤0.025). Molecular karyotyping of AML samples during therapy with hypermethylated RAD9A showed an evolving duplication of 1.8 kb on Chr16p13.3 including the PKD1 gene. Radiation, colony formation assays, cell proliferation, PCR and molecular karyotyping SNP-array experiments using generated FaDu subclones suggest that hypermethylation of RAD9A intron 2 is associated with genomic imbalances in regions with tumor-relevant genes and survival of the cells. In conclusion, this is the very first study of RAD9A intron 2 methylation in childhood cancer and Leukemia. RAD9A epimutations may have an impact on leukemia and tumorigenesis and can potentially serve as a biomarker.