HEL Cells Research Articles

Preclinical evaluation of JAK2 specific investigational oligonucleotide for the treatment of MDS/PV.

122 Background: Myeloproliferative disorders (MPD) are clonal hematopoietic stem cell malignancies with cytokine independency or hypersensitivity. Polycythemia vera (PV), an acquired MPD characterized by increased blood cell mass and hematocrit and leukocyte count, is associated with incidental myelofibrosis (MF). MDS is characterized by cytopenia and the presence of morphological dysplasia of precursor and mature bone marrow blood cells. PV and MDS leave patients at risk for progression to acute myeloid leukemia. Abnormal cytokine signaling due to an aberrant JAK2-STAT pathway has a vital role in PV and MDS pathogenesis. JAK2 mutations can result in hematologic malignancies, where hyperactive signaling of the JAK2-STAT pathway promotes tumor cell proliferation, invasion, and angiogenesis. Increased JAK2 kinase activity is observed in hematologic malignancies; somatic JAK2 V617F gain-of-function mutations are found in at least 95% of PV patients and is implicated in MDS cases. We hypothesize that preventing JAK2 transcription by antisense oligonucleotide (ASO) mediated exon masking of the JAK2 intron-exon junction will result in reduced JAK2 mRNA, and thus JAK2 protein, by providing nonsense mediated decay (NMD) in the reading frame. This may alleviate disease manifestations of hematologic malignancies like PV and MDS. Methods: This study utilized a HEL cell line harboring the V617F JAK2 gain-of-function mutation. We designed a series 19mer ASO targeting JAK2 exon-intron junctions ( JAK2 intron-exon junction providing NMD in the reading frame, steric, non-RNAse H1) and tested these at a range of concentrations. The ASOs were designed with a phosphorothioate 2’-O-methoxyethyl backbone and prioritized based on in silico binding affinity and limited off-target binding. HEL cells underwent ASO treatment (1µM) and 72-hour incubation. Results: We observed significant JAK2 protein decrease (~50%) in ASO-treated samples compared to untreated samples. JAK2 qPCR results confirmed 40-60% of target transcript. STAT5 phosphorylation status further confirmed this effect, and we report a 35% pSTAT5 reduction. Furthermore, this ASO showed limited off-target effects in silico. siRNA and CRISPR knockout lysate were used as controls. Our preclinical data support this ASO as a highly specific JAK2 agent, affecting direct levels of JAK2 as well as downstream STAT signaling. Conclusions: Around 50-60% of primary MF patients harbor the JAK2 V617F gain-of-function mutation. ASOs offer the capability to directly target JAK2 mutations with high precision and effectively reduce JAK2 protein production, without off-target kinase effects. The ability to reduce JAK2 protein may alleviate the disease burden that patients with hematologic malignancies face, resulting in a higher quality of life through prevention and treatment.

TSC22D3 as an immune-related prognostic biomarker for acute myeloid leukemia

Acute myeloid leukemia (AML) is the type of hematologic neoplasm most common in adults. Glucocorticoid-induced gene TSC22D3 regulates cell proliferation through its function as a transcription factor. However, there is no consensus on the prognostic and immunoregulatory significance of TSC22D3 in AML. In the present study, we evaluated the correlation between TSC22D3 expression, immunoinfiltration, and prognostic significance in AML. Knockdown of TSC22D3 significantly attenuated the proliferation of Hel cells and increased sensitivity to cytarabine (Ara-c) drugs. Furthermore, TSC22D3 reduced the release of interleukin-1β (IL-1β) by inhibiting the NF-κB/NLRP3 signaling pathway, thereby inhibiting macrophage polarization to M1 subtype, and attenuating the pro-inflammatory tumor microenvironment. In conclusion, this study identified TSC22D3 as an immune-related prognostic biomarker for AML patients and suggested that therapeutic targeting of TSC22D3 may be a potential treatment option for AML through tumor immune escape.

Formononetin acts synergistically with a JAK2 inhibitor to suppress growth in myeloproliferative neoplasm by inhibiting JAK/STAT3 signaling pathway

Purpose: To determine the effect of formononetin (FMNT) in the proliferation, drug resistance, and DNA damage of myeloproliferative neoplasm (MPN), and to evaluate the potential of FMNT as a therapeutic target.
 Methods: Cell viability curves and colony formation assays were used to characterize the proliferation of HEL or HEL/R cells. Flow cytometry was conducted to assess the apoptosis of HEL or HEL/R cells, while western blotting was performed to evaluate the expressions of DNA H2AX, DNA-PK, and Rad51, which are indicative of DNA damage, and the phosphorylation levels of JAK2 and STAT3 were determined.
 Results: Formononetin (FMNT) suppressed cell proliferation of HEL cells in a dose-dependent manner. It significantly reduced colony formation and promoted apoptosis of HEL cells (p < 0.05). For HEL/R cells, FMNT treatment significantly reduced cell viability, and resistance, and promoted apoptosis. Moreover, FMNT elevated H2AX levels and significantly reduced the expressions of DNA-PK and Rad51 in TG101209 (TG)-induced JAK-TKI-resistant cells (p < 0.05). Furthermore, FMNT decreased JAK2 and STAT3 phosphorylation in JAK- TKI-resistant cells.
 Conclusion: Treatment with FMNT represses proliferation, promotes apoptosis, weakens JAK-TKI resistance, and strengthens DNA damage in MPN cells by suppressing JAK/STAT3 pathway. This suggests that FMNT is a potential therapeutic candidate for the treatment of MPN.

Role of Diacylglycerol Kinases in Acute Myeloid Leukemia.

Diacylglycerol kinases (DGKs) play dual roles in cell transformation and immunosurveillance. According to cancer expression databases, acute myeloid leukemia (AML) exhibits significant overexpression of multiple DGK isoforms, including DGKA, DGKD and DGKG, without a precise correlation with specific AML subtypes. In the TGCA database, high DGKA expression negatively correlates with survival, while high DGKG expression is associated with a more favorable prognosis. DGKA and DGKG also feature different patterns of co-expressed genes. Conversely, the BeatAML and TARGET databases show that high DGKH expression is correlated with shorter survival. To assess the suitability of DGKs as therapeutic targets, we treated HL-60 and HEL cells with DGK inhibitors and compared cell growth and survival with those of untransformed lymphocytes. We observed a specific sensitivity to R59022 and R59949, two poorly selective inhibitors, which promoted cytotoxicity and cell accumulation in the S phase in both cell lines. Conversely, the DGKA-specific inhibitors CU-3 and AMB639752 showed poor efficacy. These findings underscore the pivotal and isoform-specific involvement of DGKs in AML, offering a promising pathway for the identification of potential therapeutic targets. Notably, the DGKA and DGKH isoforms emerge as relevant players in AML pathogenesis, albeit DGKA inhibition alone seems insufficient to impair AML cell viability.

Discovery of diverse sesquiterpenoids from Magnolia grandiflora with cytotoxic activities by inducing cell apoptosis

Phytochemical study of Magnolia grandiflora led to the isolation of 39 sesquiterpenoids, including 15 new compounds (1–15). Compounds 1 and 2 are discovered to be the first 13-norgermacrane type sesquiterpenoids in natural products. Compound 15 is a rare 5,6-seco-guaiane type sesquiterpene and its possible biogenic precursor is presumed to be compound 20. Subsequent structural modification for compound 28 led to 21 derivatives, among which 15 derivatives were new compounds. All compounds were tested for the inhibitory effects on three tumor cell lines, and 17 compounds were active with the IC50 values ranging from 1.91 ± 0.39 μM to 12.29 ± 1.68 μM. The structure–activity relationships implied that an α, β-unsaturated lactone group was an important active group for the cytotoxicity. Two most active compounds (19 and 29) with low toxicity on normal human liver cell line were selected for further mechanism study. Compound 29 could induce apoptosis on Colo320DM cells through influencing the key apoptotic related proteins, such as PARP, Cleaved PARP, cleaved Caspase-3, and pro-Caspase 3. In addition, compound 19 with the best cytotoxic activity on HEL cells also could induce the apoptosis in dose- and time-dependent manners. In summary, our investigation implied that compounds 19 and 29 are two new potential anti-cancer candidates for ongoing study in the future.

Immediate early response 3 gene promotes aggressive progression and autophagy of AML by negatively regulating AKT/mTOR

BackgroundImmediate early response 3 (IER3) plays a vital role in many tumors. This study aims to explore the function and mechanism of IER3 in Acute myeloid leukemia (AML). MethodsThe expression of IER3 in AML was performed by bioinformatics analysis. CCK-8 proliferation assay, flow cytometry cycle assay, clone formation assay, and tumorigenic ability were used to investigate the effect of IER3 on AML cells. Unbiased label-free quantitative proteomics and label-free quantitative phosphoproteomics analysis were performed. The regulatory relationship between SATB1(Special AT-rich sequence binding protein 1) and IER3 was investigated by Real time-PCR, Western blot, Chromatin immunoprecipitation (CHIP), and PCR. ResultsThe result indicated that the prognosis of the high IER3 expression group was significantly worse than that of the low expression group. CCK-8 assay showed that IER3 enhanced the proliferation ability. Cell cycle analysis showed IER3 could promote HL60 cells to enter the S phase of DNA synthesis from the quiescent phase. IER3 could stimulate HEL cells to enter mitosis. Clone-formation experiments suggested that IER3 enhanced clonogenic ability.IER3 promoted the tumorigenesis of AML. Further experimental investigation revealed that IER3 promoted autophagy and induced the occurrence and development of AML by negatively regulating the phosphorylation activation of AKT/mTOR pathway. SATB1 was found to bind to the promoter region of IER3 gene and negatively regulate its transcription. ConclusionIER3 could promote the development of AML and induce autophagy of AML cells by negatively regulating the phosphorylation and activation of AKT/mTOR. By the way, SATB1 may negatively target regulates IER3 transcription.

Combined inhibition of XIAP and autophagy induces apoptosis and differentiation in acute myeloid leukaemia.

Perturbations in autophagy, apoptosis and differentiation have greatly affected the progression and therapy of acute myeloid leukaemia (AML). The role of X-linked inhibitor of apoptosis (XIAP)-related autophagy remains unclear in AML therapeutics. Here, we found that XIAP was highly expressed and associated with poor overall survival in patients with AML. Furthermore, pharmacologic inhibition of XIAP using birinapant or XIAP knockdown via siRNA impaired the proliferation and clonogenic capacity by inducing autophagy and apoptosis in AML cells. Intriguingly, birinapant-induced cell death was aggravated in combination with ATG5 siRNA or an autophagy inhibitor spautin-1, suggesting that autophagy may be a pro-survival signalling. Spautin-1 further enhanced the ROS level and myeloid differentiation in THP-1 cells treated with birinapant. The mechanism analysis showed that XIAP interacted with MDM2 and p53, and XIAP inhibition notably downregulated p53, substantially increased the AMPKα1 phosphorylation and downregulated the mTOR phosphorylation. Combined treatment using birinapant and chloroquine significantly retarded AML progression in both a subcutaneous xenograft model injected with HEL cells and an orthotopic xenograft model injected intravenously with C1498 cells. Collectively, our data suggested that XIAP inhibition can induce autophagy, apoptosis and differentiation, and combined inhibition of XIAP and autophagy may be a promising therapeutic strategy for AML.

Abstract 6285: Preclinical efficacy of CDK7 inhibitor-based combinations in cellular models of advanced myeloproliferative neoplasms (MPN)

Abstract Advanced MPN are characterized by driver mutations in JAK2, c-MPL or calreticulin (CALR) gene, with hyperactive JAK-STAT5/3 and NFkB signaling. Co-occurrence of mutations in chromatin/transcriptional regulators, including DNMT3A, TET2, ASXL1, EZH2, SRSF2, RUNX1 and TP53, creates the dysregulated transcriptome, which is responsible for the increased growth, survival and therapy-refractoriness in MPN blastic phase (BP). Treatment with JAK inhibitors (JAKis), e.g., ruxolitinib, venetoclax and hypomethylating agents are ineffective in improving survival in MPN-BP. CDK7 is the crucial catalytic component of the general transcript initiation factors TFIIH, which is recruited to the transcription start-sites along with RNA-pol II (RNAP2) to initiate transcription. Complexed with cyclin H, CDK7 phosphorylates CDK1, 2, 4 and 6, as well as serine 5 and 7 in the C terminal domain (CTD) of RNAP2, thus facilitating transcript initiation. CDK7 also phosphorylates CDK9, which in turn phosphorylates serine 2 of the CTD of RNAP2, and phosphorylates and inactivates the negative elongation factors, thus enabling productive mRNA transcript elongation of growth and survival promoting oncogenes. In MPN-BP, these include RUNX1, Bcl-xL, MCL1, CDK4/6, PIM1 and MYC. CRISPR-gRNA dependency-screen (DepMap) demonstrated that CDK7 is a dependency in the Advanced-AML cell lines, SET2 and HEL cells. Present studies demonstrate that treatment with ATP-competitive, covalent CDK7 inhibitors (CDK7i) SY-1365, and its clinical grade counterpart SY-5609, dose-dependently inhibits cell cycle, growth and induces lethality in SET2 and HEL as well as patient-derived (PD), CD34+ MPN-sAML cells. ChIP-Seq analysis showed that SY-1365 treatment caused log2 fold-reduction in p-Ser2-RNAP2 occupancy on the DNA of MYC, PIM1 and MCL1 genes in HEL cells. RNA-Seq analysis showed that SY1365 treatment caused negative enrichment of gene-sets of MYC and E2F targets, as well as inflammatory response, IL6-JAK-STAT3 and mTORC1 signaling. SY-1365 treatment also dose-dependently depleted protein expressions of p-Ser5 RNAP2, p-Ser2 RNAP2, c-Myc, c-Myb, RUNX1, PU.1, MCL1 and Bcl-xL, while inducing cleavage of PARP protein. As compared to the parental JAKi-sensitive, in vitro generated JAKi-persister/resistant (P/R) SET2/RuxP cells were significantly more sensitive to SY-1365-induced apoptosis. Co-treatment with CDK7i and ruxolitinib or the BET inhibitor OTX015 was synergistically lethal in PD MPN-BP cells (n=7). In an aggressive xenograft model of HEL-GFP/Luc cells in NSG mice, compared to the vehicle control, monotherapy with SY5609 significantly reduced the MPN sAML burden and improved survival of the NSG mice without causing host toxicity. These findings demonstrate promising activity and support the rationale to further evaluate the in vivo efficacy of CDK7i-based combinations against advanced MPN. Citation Format: Christopher P. Mill, Warren C. Fiskus, Christine E. Birdwell, John A. Davis, Kaberi Das, Kapil N. Bhalla. Preclinical efficacy of CDK7 inhibitor-based combinations in cellular models of advanced myeloproliferative neoplasms (MPN) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6285.

Stable Expression of Coagulation Factors by RPS6 Promoter

To screen better promoters and provide more powerful tools for basic research and gene therapy of hemophilia. Bioinformatics methods were used to analyze the promoters expressing housekeeping genes with high abundance, so as to select potential candidate promoters. The GFP reporter gene vector was constructed, and the packaging efficiency of the novel promoter was investigated with EF1 α promoter as control, and the transcription and activities of the reporter gene were investigated too. The activity of the candidate promoter was investigated by loading F9 gene. The most potential RPS6 promoter was obtained by screening. There was no difference in lentiviral packaging between EF1 α-LV and RPS6-LV, and their virus titer were consistent. In 293T cells, the transduction efficiency and mean fluorescence intensity of RPS6pro-LV and EF1 αpro-LV were proportional to the lentiviral dose. The transfection efficiency of both promoters in different types of cells was in the following order: 293T>HEL>MSC; Compared with EF1 αpro-LV, RPS6pro-LV could obtain a higher fluorescence intensity in MSC cells, and RPS6pro-LV was more stable in long-term cultured HEL cells infected with two lentiviruses respectively. The results of RT-qPCR, Western blot and FIX activity (FIX∶C) detection of K562 cell culture supernatant showed that FIX expression in the EF1 α-F9 and RPS6-F9 groups was higher than that in the unloaded control group, and there was no significant difference in FIX expression between the EF1 α-F9 and RPS6-F9 groups. After screening and optimization, a promoter was obtained, which can be widely used for exogenous gene expression. The high stability and viability of the promoter were confirmed by long-term culture and active gene expression, which providing a powerful tool for basic research and clinical gene therapy of hemophilia.

2-Aminopyrimidine derivatives as selective dual inhibitors of JAK2 and FLT3 for the treatment of acute myeloid leukemia

Dual inhibitors of JAK2 and FLT3 can synergistically control the development of acute myeloid leukemia (AML), and overcome secondary drug resistance of AML that is associated with FLT3 inhibition. We therefore designed and synthesized a series of 4-piperazinyl-2-aminopyrimidines as dual inhibitors of JAK2 and FLT3, and improved their selectivity for JAK2. Screening cascades revealed that compound 11r exhibited inhibitory activity with IC50 values of 2.01, 0.51, and 104.40 nM against JAK2, FLT3, and JAK3, respectively. Compound 11r achieved a high selectivity for JAK2 at a ratio of 51.94, and also showed potent antiproliferative activity in HEL (IC50 = 1.10 μM) and MV4-11 (IC50 = 9.43 nM) cell lines. In an in vitro metabolism assay, 11r exhibited moderate stability in human liver microsomes (HLMs), with a half-life time of 44.4 min, and in rat liver microsomes (RLMs), with a half-life of 143 min. In pharmacokinetic studies, compound 11r showed moderate absorption (Tmax = 5.33 h), with a peak concentration of 38.7 ng/mL and an AUC of 522 ng h/mL in rats, and an oral bioavailability of 25.2%. In addition, 11r induced MV4-11 cell apoptosis in a dose-dependent manner. These results indicate that 11r is a promising selective JAK2/FLT3 dual inhibitor.

A JAK tyrosine kinase and pseudokinase Co-inhibition strategy combines enhanced potency and on-demand activation

Janus tyrosine kinase (JAK) inhibitors have been on the market for several years, but their use is limited by drug resistance and intolerable side effects. Herein, we propose a novel strategy of JAK tyrosine kinase (TK) and pseudokinase (PK) domain co-inhibition system to consolidate robust JAK inhibition and on-demand activation. A photoexcited prodrug PAT-SIL-TG-1&AT exhibits the synergy effects of TK-PK co-inhibition and enable the spatiotemporal control of JAK2 signaling. The hypoxia-activated prodrug HAT-SIL-TG-1&AT significantly inhibited HEL cells proliferation and downregulated phosphorylated STAT3/5 under hypoxic conditions. Importantly, HAT-SIL-TG-1&AT showed synergistic antitumor effects and selectively inhibited the JAK-STAT signaling in tumor tissues in vivo. This work demonstrates a viable solution to achieve superior JAK2 inhibition, and provides an inspiration for other kinases containing PK domain.

FLI1 accelerates leukemogenesis through transcriptional regulation of pyruvate kinase-L/R and other glycolytic genes.

In cancer cells, multiple oncogenes and tumor suppressors control glycolysis to sustain rapid proliferation. The ETS-related transcription factor Fli1 plays a critical role in the induction and progression of leukemia, yet, the underlying mechanism of this oncogenic event is still not fully understood. In this study, RNAseq analysis of FLI1-depleted human leukemic cells revealed transcriptional suppression of the PKLR gene and activation of multiple glycolytic genes, such as PKM1/2. Pharmacological inhibition of glycolysis by PKM2 inhibitor, Shikonin, significantly suppressed leukemic cell proliferation. FLI1 directly binds to the PKLR promoter, leading to the suppression of this inhibitor of glycolysis. In accordance, shRNA-mediated depletion of PKLR in leukemic HEL cells expressing high levels of FLI1 accelerated leukemia proliferation, pointing for the first time to its tumor suppressor function. PKLR knockdown also led to downregulation of the erythroid markers EPOR, HBA1, and HBA2 and suppression of erythroid differentiation. Interestingly, silencing of PKLR in HEL cells significantly increased FLI1 expression, which was associated with faster proliferation in culture. In FLI1-expressing leukemic cells, lower PKLR expression was associated with higher expression of PKM1 and PKM2, which promote aerobic glycolysis. Finally, injection of pyruvate, a known inhibitor of glycolysis, into leukemia mice significantly suppressed leukemogenesis. These results demonstrate that FLI1 promotes leukemia in part by inducing glycolysis, implicates PKLR in erythroid differentiation, and suggests that targeting glycolysis may be an attractive therapeutic strategy for cancers driven by FLI1 overexpression.

Novel chemical-structure TPOR agonist, TMEA, promotes megakaryocytes differentiation and thrombopoiesis via mTOR and ERK signalings

BackgroundNon-peptide thrombopoietin receptor (TPOR) agonists are promising therapies for the mitigation and treatment of thrombocytopenia. However, only few agents are available as safe and effective for stimulating platelet production for thrombocytopenic patients in the clinic. PurposeThis study aimed to develop a novel small molecule TPOR agonist and investigate its underlying regulation of function in megakaryocytes (MKs) differentiation and thrombopoiesis. MethodsA potential active compound that promotes MKs differentiation and thrombopoiesis was obtained by machine learning (ML). Meanwhile, the effect was verified in zebrafish model, HEL and Meg-01 cells. Next, the key regulatory target was identified by Drug Affinity Responsive Target Stabilization Assay (DARTS), Cellular Thermal Shift Assay (CETSA), and molecular simulation experiments. After that, RNA-sequencing (RNA-seq) was used to further confirm the associated pathways and evaluate the gene expression induced during MK differentiation. In vivo, irradiation (IR) mice, C57BL/6N-TPORem1cyagen (Tpor-/-) mice were constructed by CRISPR/Cas9 technology to examine the therapeutic effect of TMEA on thrombocytopenia. ResultsA natural chemical-structure small molecule TMEA was predicted to be a potential active compound based on ML. Obvious phenotypes of MKs differentiation were observed by TMEA induction in zebrafish model and TMEA could increase co-expression of CD41/CD42b, DNA content, and promote polyploidization and maturation of MKs in HEL and Meg-01 cells. Mechanically, TMEA could bind with TPOR protein and further regulate the PI3K/AKT/mTOR/P70S6K and MEK/ERK signal pathways. In vivo, TMEA evidently promoted platelet regeneration in mice with radiation-induced thrombocytopenia but had no effect on Tpor-/- and C57BL/6 (WT) mice. ConclusionTMEA could serve as a novel TPOR agonist to promote MKs differentiation and thrombopoiesis via mTOR and ERK signaling and could potentially be created as a promising new drug to treat thrombocytopenia.

Evaluation of Chiral Organosulfur Compounds on Their Activity against the Malaria Parasite Plasmodium falciparum.

Malaria is one of the deadliest tropical diseases, especially causing havoc in children under the age of five in Africa. Although the disease is treatable, the rapid development of drug resistant parasites against frontline drugs requires the search for novel antimalarials. In this study, we tested a series of organosulfur compounds from our internal library for their antiplasmodial effect against Plasmodium falciparum asexual and sexual blood stages. Some active compounds were also obtained in enantiomerically pure form and tested individually against asexual blood stages of the parasite to compare their activity. Out of the 23 tested compounds, 7 compounds (1, 2, 5, 9, 15, 16, and 17) exhibited high antimalarial activity, with IC50 values in the range from 2.2 ± 0.64 to 5.2 ± 1.95 µM, while the other compounds showed moderate to very low activity. The most active compounds also exhibited high activity against the chloroquine-resistant strain, reduced gametocyte development and were not toxic to non-infected red blood cells and Hela cells, as well as the hematopoietic HEL cell line at concentrations below 50 µM. To determine if the enantiomers of the active compounds display different antimalarial activity, enantiomers of two of the active compounds were separated and their antimalarial activity compared. The results show a higher activity of the (-) enantiomers as compared to their (+) counterparts. Our combined data indicate that organosulfur compounds could be exploited as antimalarial drugs and enantiomers of the active compounds may represent a good starting point for the design of novel drugs to target malaria.

The indispensable role of the RNA helicase DDX5 in tumorigenesis induced by the myeloproliferative neoplasm-associated JAK2V617F mutant

A point mutation (V617F) in the Janus kinase 2 (JAK2) gene results in the production of disorderly activated tyrosine kinase, which causes myeloproliferative neoplasms (MPN). We herein demonstrated that the RNA helicase DDX5 was highly expressed at the mRNA and protein levels through the activation of signal transducer and activator of transcription 5 (STAT5) in Ba/F3 cells expressing a JAK2V617F mutant and erythropoietin receptor (V617F/EpoR cells) and MPN patient-derived HEL cells. A treatment with the JAK1/2 inhibitor, ruxolitinib and STAT5 inhibitor, pimozide significantly inhibited DDX5 mRNA expression and enhanced the degradation of DDX5 in these cells, suggesting that the JAK2V617F mutant positively regulates DDX5 mRNA expression and DDX5 protein stability by activating STAT5. The knockdown of DDX5 specifically inhibited the activation of mechanistic target of rapamycin (mTOR) in V617F/EpoR cells and HEL cells and significantly suppressed the proliferation of these cells. Furthermore, the knockdown of DDX5 markedly suppressed tumorigenesis, splenomegaly, and liver hypertrophy caused by an inoculation of V617F/EpoR cells in nude mice. Collectively, these results revealed that JAK2V617F exhibits transforming activity by inducing the expression of DDX5 in a STAT5-dependent manner, indicating the potential of the JAK2V617F/STAT5/DDX5 axis as a therapeutic target in the treatment of MPN.

Tyrosine Phosphorylation of CARM1 Promotes Its Enzymatic Activity and Alters Its Target Specificity in Myeloid Neoplasms with Hyperactivated JAK2

Introduction Coactivator-associated arginine methyltransferase 1 (CARM1) catalyzes the asymmetric dimethylation of arginine residues in histone and non-histone substrates, including transcription factors, RNA splicing factors, and the SWI/SNF chromatin remodeling complex. CARM1 has emerged as an important target in acute myeloid leukemia. We have previously shown that Janus kinase 2 (JAK2) affects the activity of epigenetic modifiers. A V617F mutation in JAK2 genes leads to its hyperactivation. This mutation is a common hallmark of the BCR/ABL1-negative myeloproliferative neoplasms, and also found in patients with acute myeloid leukemia and myelodysplastic syndrome. Here, we find that hyperactivated JAK2, mediated by biallelic V617F mutation, triggers CARM1 phosphorylation, increasing its methyltransferase activity and altering its target specificity. Methods We generated two phospho-tyrosine specific antibodies against CARM1 tyrosine-149 (Y139) and -334 (Y334), and two asymmetric dimethylation-arginine specific antibodies against RUNX1 arginine-223 (R223) and -319 (R319). We also generated isogenic HEL cells carrying homozygous CARM1 non-phosphorylatable mutation (Y149F or Y334F single, or Y149F/Y334F double mutation), using the CRISPR/Cas9 nuclease system. Results We first performed cell-free in vitro kinase assays using recombinant CARM1 protein and JAK2 kinase, and mass spectrometry analysis, which identified Y139 and Y334 as the sites of JAK2 phosphorylation in CARM1. We next compared the levels of CARM1-Y149 and -Y334 phosphorylation in 15 myeloid leukemia cell lines and normal human CD34+ cord blood cells. HEL and UKE-1 cells, that harbor bi-allelic JAK2-V617F mutation, showed the highest level of phosphorylated CARM1-Y149 and -Y334, while SET2 cells with mono-allelic JAK2-V617F mutation and the other cell lines showed abundant CARM1 protein but a lower relative amount of phosphorylated CARM1 protein. We found that the JAK2 activation, through transphosphorylation by JAK2 and TYK2, enhances tyrosine kinase activity against CARM1 using HEL cells with knockout of either JAK1 or TYK2. Having previously shown that CARM1 methylates RUNX1 at R223, in an immunoprecipitation assay, WT-CARM1 was able to pull down RUNX1, while the non-phosphorylatable CARM1 mutants were not. Using mass spectrometry and an in vitro methylation assay, we confirmed R223 and R319 as CARM1 target sites. We examined non-phosphorylatable CARM1 mutation knock-in HEL cells and found that mutation of either site reduces the asymmetric dimethylation of RUNX1 as well as known substrates (BAF155 and PABP1). We observed that CARM1 Y149F/Y334F mutant-expressing HEL cells reduced cell proliferation with G2/M cell cycle arrest and an increase in apoptosis. To identify the transcription networks regulated by CARM1 phosphorylation, we performed RNA-seq on WT-CARM1 and double CARM1 Y149F/Y334F mutant-expressing HEL cells. Gene set enrichment analysis identified that gene sets associated with G2/M cell cycle progression and anti-apoptosis were significantly downregulated in Y149F/Y334F mutation knock-in HEL cells. We next performed ChIP-seq analyses using antibodies against total RUNX1 and against asymmetrically dimethylated R319-RUNX1 in HEL cells expressing knock-in of CARM1 non-phosphorylatable mutation. CARM1 non-phosphorylatable mutation decreased the signals of dimethylated RUNX1-R319 within 5 kb of the transcription start sites for RUNX1 target genes, with less effect on total RUNX1. We next assessed the efficacy of the combination with CARM1 inhibitor (EPZ025654) and JAK2 inhibitor (ruxolitinib). Dual targeting inhibition of JAK2 and CARM1 showed a synergistic inhibition effect on HEL and UKE-1 cells (with abundant phosphor-CARM1), but only an additive effect on SET2 cells (with less amount of phosphor-CARM1). Conclusion CARM1 phosphorylation mediated by hyperactivated JAK2 regulates its methyltransferase activity and is required for the maximal proliferation of myeloid neoplasms. We also demonstrated that dual targeting of JAK2 and CARM1 is more effective than monotherapy in cell lines with phosphor-CARM1. Thus, our results reveal a novel role for CARM1 phosphorylation in myeloid neoplasms.

Synthetic Lipid Nanoparticles Actively Target Acute Myeloid Leukemia (AML) Cells and Induce Ferroptosis through Decreased Expression of Glutathione Peroxidase 4

Introduction: Relapsed and refractory AML patients have a 30% 5-year overall survival (SEER 2012-2018). We found that certain cancer cells that are highly dependent on cholesterol uptake and express the high-affinity receptor for cholesterol-rich high-density lipoproteins (HDL), scavenger receptor type B-1 (SR-B1), also highly express the redox enzyme, glutathione peroxidase 4 (GPX4) (Rink et al 2020). GPX4 has been shown to detoxify lipid peroxides in cancer cells and prevent cell death through ferroptosis, an iron-dependent redox form of cell death. Using a synthetic lipid nanoparticle (Au-LNP) that actively targets SR-B1, we showed modulation of cancer cell cholesterol uptake and metabolism is accompanied by a reduction in GPX4 expression. As a result, Au-LNP therapy kills these cells via ferroptosis. In AML, high GPX4 expression is associated with worse overall survival (Akiyama et al. Blood 2021). Therefore, we hypothesized that high SR-B1 expression would enable Au-LNP to target AML cells, modulate cell cholesterol, and reduce GPX4 resulting in AML cell death by ferroptosis. Methods: GEPIA is an interactive web server that houses the RNA sequencing expression data from the TCGA and the GTEx projects. We selected the LAML database and interrogated the survival data using the top 25% and bottom 25% SR-B1 expression as cutoffs. The cell lines MV4-11 (biphenotypic B-myelomonocytic AML with translocation t(4;11) and FLT3-ITD mutation), HEL (erythroblast AML), and U937 (promonocytic AML) were used for these studies. Au-LNPs were synthesized by surface-functionalizing 5nm gold (Au) nanoparticles with apolipoprotein A-I and phospholipids. SR-B1 and GPX4 levels were measured by western blot and flow cytometry. Cell viability was measured using the MTS assay, annexin V/propidium iodide staining, and a colony forming assay. Colonies were counted by an automated microscope and analyzed with Fiji. Lipid oxidation levels were measured by a C11 BODIPY flow cytometry assay. Surface biomarker changes were measured by flow cytometry. Primary AML samples were obtained by collecting the buffy coat from peripheral blood samples from 2 patients with AML and isolating CD34 positive blasts (IRB# STU00208941). Results: In the LAML database in GEPIA, we found a significant survival detriment with high SR-B1 expression (75th percentile) when compared with low SR-B1 expression (25th percentile; p = 0.023; hazard ratio = 2.5) (Figure 1). SR-B1 expression was observed in all three AML cell lines tested (MV4-11, HEL, U937). Au-LNPs potently induced cell death in each cell line, with IC50s for MV4-11 (4.7 nM), HEL (0.8 nM), and U937 (4.6 nM) that were much lower than with cytarabine (535 nM, 259 nM, and 96 nM, respectively). In the colony forming assays, Au-LNPs significantly decreased MV4-11 colony counts compared with PBS control. By contrast, β-Hydroxy β-methylglutaryl-CoA (HMG-CoA) inhibitors, such as atorvastatin and rosuvastatin, had no effect on cell viability, with concentrations up to 50 µM. Au-LNPs reduced GPX4 expression in MV4-11 and HEL cells, leading to the accumulation of lipid peroxides by C11 BODIPY assay. Au-LNPs-induced cell death was prevented by the addition of the lipophilic antioxidant ferrostatin-1 and the iron chelator deferoxamine (DFO), supporting the hypothesis that Au-LNP-induced cell death occurred by ferroptosis. Low dose Au-LNPs increased expression of CD11c (dendritic cell marker), CD16 (monocyte marker), and CD24 (neutrophil marker) on MV4-11 cells, suggesting an increase in myeloid differentiation. Finally, we isolated CD34+ blasts from peripheral blood samples of two AML patients. Both patients’ blasts expressed SR-B1 as detected by flow cytometry, and Au-LNPs showed significant cell killing in both patient samples. Conclusion: We have demonstrated that Au-LNP target SR-B1, expressed by both AML cell lines and in primary AML samples, and induce cell death by ferroptosis resulting from depletion of GPX4. Future studies targeting the cholesterol scavenger receptor SR-B1 and investigating relevant resistance pathways and opportunities for synergy with available agents are indicated. Figure 1. Overall survival of AML patients in the LAML database stratified by scavenger receptor B1 expression. (Tang, Z. et al. (2017) GEPIA: a web server for cancer and normal gene expression profiling and interactive analyses. Nucleic Acids Res, 10.1093/nar/gkx247) Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

SLFN11 Is a Potential Biomarker and Modulator of Treatment Response in AML

Introduction: Despite recent advances in treatment for acute myeloid leukemia (AML), AML remains a deadly disease, with approximately 70% of patients succumbing to the disease within five years of diagnosis (SEER database 2021). Intensive induction chemotherapy carries a risk of morbidity and mortality, making it a risky treatment for patients who are unlikely to achieve a remission with DNA damaging-based treatments. Hypomethylating agents (HMAs) such as azacitidine (AZA) and decitabine (DAC) are now frequently used in AML treatment as less intensive treatment options. Thus, biomarkers to predict response to DNA damaging agents in AML are needed. We recently found a significant association between AML patient survival and high expression of Schlafen11 (SLFN11), a dynamically inducible key effector of the DNA replication stress response. SLFN11 has been shown to be associated with response to certain chemotherapy agents in some solid tumors in vitro and in vivo (Conteduca et. al. JCO 2019; Shee et. al. PLoS One 2019), but its importance in leukemia has not been well defined. It is also unknown whether SLFN11 levels affect response to HMAs. We hypothesized that SLFN11 can be used as a predictive biomarker for treatment response to DNA-damaging agents in AML but that response to HMAs would be independent of HMAs. We also hypothesized that identifying pathways upregulated in SLFN11-low cells could lead to novel combination therapies to increase sensitivity to chemotherapeutics. Methods: The human leukemia cell lines HEL and U937 cells were used for these studies. U937- and HEL-SLFN11 knockout (SLFN11KO) cells were generated using CRISPR-Cas9 gene editing technology. Cell viability was assessed by WST-1 reagent assay. SLFN11 and other protein levels were measured by Western blotting. Results: SLFN11 protein expression was readily observed by immunoblotting of HEL and U937 cell lysates. U937- and HEL-SLFN11KO cells were incubated with increasing concentrations of cytarabine (AraC), which is the backbone of most chemotherapy regimens currently used in AML treatment. Both U937- and HEL-SLFN11KO cells were more resistant to AraC than their respective control cells. To examine the mechanism of resistance in U937-SLFN11KO cells, SLFN11KO and control cells were treated with AraC for up to 8 hrs, and lysates were subjected to immunoblotting for various DNA damage response proteins. There were higher baseline levels of multiple DNA damage-related proteins (including Chk1 and Chk2) in the SLFN11KO cells. There was also prolonged activation of the DNA damage pathway in U937-SLFN11KO cells, as evidenced by phosphorylation of the ATR substrate Chk1 (Ser345) through 8 hours, whereas phospho-Chk1 levels decreased in control cells by 8 hours. Interestingly, this prolonged activation occurred without a concomitant increase in γH2AX (a marker of DNA damage and double strand breaks). Because U937-SLFN11KO cells demonstrated a more pronounced activation of the ATR/Chk1 pathway in response to AraC, we subjected U937-SLFN11KO and control cells to increasing concentrations of AraC in combination with a small dose (0.3 μM) of the ATR inhibitor VE822. Notably, ATR inhibition restored sensitivity of SLFN11KO cells to AraC. These results suggest that SLFN11KO cells are resistant to DNA damaging agents via upregulation and activation of DNA repair proteins, and that adding inhibitors of these proteins can decrease resistance to chemotherapy agents in SLFN11KO cells. In contrast, SLFN11KO cells remained sensitive to AZA and DAC, suggesting that HMAs may be preferable to AraC for patients with SLFN11-low AML. Conclusions: Low SLFN11 levels predict poor response to standard chemotherapy agents (i.e. AraC) in AML cells, suggesting that SLFN11 may prove to be an important predictive biomarker for AML patients. In addition, SLFN11-low AML cells have an increased reliance on DNA repair pathways, and inhibition of DNA repair proteins such as ATR may enhance responses to AraC in these cells. This raises the possibility of patient selection based on SLFN11 levels for specific combination approaches for the treatment of AML. Figure 1. Kaplan Meier survival curves of AML patients in the TCGA (LAML) database with high (top 50th percentile) or low (bottom 50th percentile) SLFN11 expression using UCSC Xena (Goldman et. al. Nat Biotechnol 2020). Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

UBASH3B knockout Downregulates STAT1 Mediated Interferon Signalling and Megakaryocyte Related Genes in a JAK2 V617F Positive MPN Model

Interferon (IFN) signalling via STAT1 plays an important role in the pathogenesis and treatment of myeloproliferative neoplasms (MPN). Single cell sequencing approaches have implicated megakaryocyte (MKC) biased stem cells with enhanced intrinsic activation of IFN signalling pathways in MPN patients as critical mediators of disease initiation and maintenance. UBASH3B is a histidine phosphatase and has been identified as a potential regulator of malignant haematopoiesis and as a mediator of JAK/STAT1 IFN signalling in lymphoid cells. RNA sequencing (RNA-seq) data in JAK2 V617F positive UKE-1, HEL (both own lab) and SET2 (GSE91062) cells demonstrate that UBASH3B is significantly downregulated following administration of the JAK inhibitor ruxolitinib (RUX). We observed that UBASH3B expression in CD34+ cells in patients with primary myelofibrosis (GSE53482) is significantly enhanced but reduced in patients with essential thrombocythaemia(ET) and polycythaemia (GSE103237) and hypothesized that UBASH3B may contribute to MPN pathogenesis. UKE-1 is a JAK2 V617F positive primitive haematopoietic cell line derived from an ET patient. It shows features of intrinsic IFN signalling and expresses genes related to MKC development and differentiation providing a useful model of MPN stem cell behaviour. CRISPR-Cas9 mediated KO of UBASH3B was performed in UKE-1 cells. A ribonucleoprotein complex of guide RNA and Cas9 protein was nucleofected into cells. Individual colonies were assessed for UBASH3B status using western blotting and Sanger sequencing. Four distinct, healthy KO clones were established for the UKE-1 cell line and compared to the original wildtype cell population. RNA-seq and phospho-proteomic analysis were undertaken in two KO clones. Extracted RNA was sequenced on Illumina platforms with a minimum of 45 million individual paired reads per sample. Differential gene expression was analysed using DESEQ2. Whilst, matched cell lysates underwent tandem mass tagging, high pH fractionation, and phospho-peptide enrichment was undertaken prior to liquid chromatography-mass spectrometry for total and phospho-proteomic analysis. RNA-seq and total proteomic analysis were consistent in demonstrating a significant downregulation of STAT1 (Log2FC-0.79, FDR 9.03E-49) and STAT2 (Log2FC -0.70, FDR 9.05E-20) with over-enrichment analysis identifying downregulation of type 1 IFN signalling pathways and pathways related to MKC and platelet function as shown in Figure 1. The significant downregulation of genes associated with platelet function, MKC development and differentiation included GATA1, GP1BA, ITGA2B, SELP, VWF and genes identified as mediators of fibrosis in MPN as demonstrated in Figure 2. Targeted real time PCR for STAT1, GP1BA and LTPB1 confirmed the same transcriptional patterns were present in both additional knockout clones. Knockout clones show a diminished response to exogenous IFN alpha administration consistent with downregulation of these pathways. Consistently reduced proliferation is observed in the KO clones by a variety of methodologies. Comparison to the transcriptional response in RUX treated UKE-1 cells demonstrates that at 24 hours post RUX treatment significant STAT1 downregulation is observed but not accompanied by the same pattern of down-regulation of MKC and fibrosis associated genes. The same was seen in cells persistently treated with high dose RUX for greater than 5 weeks. These results show a significant downregulation of IFN signalling in UBASH3B KO cells linked to a significant downregulation of MKC associated genes further highlighting the link observed in MPN stem cells. They implicate UBASH3B as a potential mediator of IFN signalling in a JAK2 V617F context and support a hypothesis that downregulating IFN signalling in MPN stem cells may have disease modifying potential by altering differentiation and pro-fibrotic potential. RUX treatment downregulates STAT1 without activating the same transcriptional programme associated with MKC gene downregulation and this may help explain the limited disease modifying effect of RUX observed in patients. This work has generated an important KO cell line model which will permit the molecular characterisation of differential STAT1/IFN pathway activation and allow further study to understand the regulation of MKC differentiation and pro-fibrotic gene transcription observed in MPN stem cells. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Comprehensive Profiling of Clinical JAK2 Inhibitors in Myeloproliferative Neoplasms

Since the approval of ruxolitinib (JAK1/JAK2) in 2011, two additional inhibitors, fedratinib (JAK2/FLT3) and pacritinib (JAK2/FLT3/IRAK1/CSF1R), have received FDA approval for myelofibrosis, and a new drug application for a fourth inhibitor, momelotinib (JAK1/JAK2/ACVR1/TBK1) has been submitted in June 2022. Although all four inhibitors potently suppress JAK2, the targeting of additional auxiliary targets may contribute to distinct responses observed in the clinic. Here, we take a comprehensive approach to assess the unique proliferative, transcriptional, and signaling pathways altered by these four JAK2 inhibitors in myeloproliferative neoplasms (MPN). In three human JAK2 mutant leukemic cell lines (HEL, UKE-1, SET2) and murine Ba/F3 EpoR-JAK2VF cells, ruxolitinib consistently showed the lowest IC50. Following, CD34+ hematopoietic stem/progenitor cells (HSPCs) from 8 MPN patients and 3 healthy donors were seeded for colony formation assays. Erythroid colonies were highly sensitive to ruxolitinib, fedratinib, and pacritinib inhibition. In contrast, momelotinib demonstrated greater relative sparing of erythroid colonies, consistent with clinical anemia amelioration, albeit potentially via a ACVR1/ALK2 and hepcidin-independent manner. Overall, greatest potency was demonstrated by pacritinib ex vivo, followed by ruxolitinib, fedratinib, and momelotinib. These findings were somewhat distinct from in vitro assays, likely reflecting additional underlying biology of human HSPCs. We then performed high dimensional mass cytometry to assess signaling changes at single cell resolution across different cell populations in 9 MPN patient samples and 3 healthy donors treated ex vivo with JAK inhibitors. We observed potent suppression of pSTAT1, pSTAT3, and pSTAT5 in CD34+ cells, with the 4 JAK2 inhibitors exerting similar degrees of inhibition. In B cells, pacritinib suppressed IκBα, pERK, and pCREB levels, while momelotinib inhibited pTBK1. Similar observations were noted in T cells, which in addition exhibited suppression of pSTAT1 and pSTAT3. These findings indicate distinct suppression responses across specific cell types and further highlight nuances beyond sole suppression of JAK-STAT signaling by these inhibitors. RNA-sequencing of HEL cells treated with the 4 JAK2 inhibitors also identified unique alteration of expression profiles and pathways (Fig. A). Differential gene expression (DEG) analysis revealed pacritinib inducing the greatest transcriptional alterations after 4 hours (3,121 genes) and ruxolitinib (1,969 genes) following 48 hours treatment duration. Only 82 and 103 DEGs relative to control were shared across the 4 JAK2 inhibitor treatment at 4 and 48 hours, respectively. Additional pathway analysis revealed at least 3 out of 4 inhibitors demonstrating capacity to suppress 1) JAK-STAT signaling, 2) inflammatory response, and 3) TNF signaling via NFκB pathways. Upregulation of multiple metabolic pathways was observed such as oxidative phosphorylation by momelotinib and pacritinib, potentially reflecting adaptive transcriptional feedback, as Seahorse Mito Stress assays revealed strongest suppression of oxygen consumption rate of PBMCs mediated by pacritinib and fedratinib. Lastly, we compared the in vivo potency of JAK2 inhibitors using patient-derived xenograft (PDX) models (Fig. B). Engrafted immunodeficient NSGS mice were treated with JAK2 inhibitors for 4 weeks across 2 sets of PDX experiments with CD34+ cells from unique JAK2 and CALR mutant donors. Pacritinib demonstrated greatest potency in reduction of peripheral blood and bone marrow human CD45+ leukemic cells, followed by ruxolitinib, and fedratinib/momelotinib. Vehicle treated mice were moribund by week 3 of treatment and survival was significantly prolonged with all JAK2 inhibitor treatments, with all animals surviving in the pacritinib treatment group after 4 weeks of treatment at end point. Despite all four JAK2 inhibitors demonstrating affinity in suppressing JAK-STAT signaling, our assays reveal varying degrees of alteration of transcriptional, proteomic, and metabolic profiles. In conjunction with comparative assessment of in vivo activity in PDX models, these findings provide insights with potential relevance to distinct responses observed with these inhibitors in the clinic and may help guide the use of specific JAK2 inhibitors in combination studies. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal