Growth Inhibition Of Leukemia Cells Research Articles

A novel α,β-unsaturated ketone inhibits leukemia cell growth as PARP1 inhibitor.

Leukemia is a malignant disease of the hematopoietic system, in which clonal leukemia cells accumulate and inhibit normal hematopoiesis in the bone marrow and other hematopoietic tissues as a result of uncontrolled proliferation and impaired apoptosis, among other mechanisms. In this study, the anti-leukemic effect of a compound (SGP-17-S) extracted from Chloranthus multistachys, a plant with anti-inflammatory, antibacterial and anti-tumor effects, was evaluated. The effect of SGP-17-S on the viability of leukemic cell was demonstrated by MTT assay, cell cycle, and apoptosis were assessed by flow cytometry using PI staining and Annexin V/PI double staining. Combinations of network pharmacology and cellular thermal shift assay (CETSA) with western blot were used to validate agents that act on leukemia targets. The results showed that SGP-17-S inhibited the growth of leukemia cells in a time- and dose-dependent manner. SGP-17-S blocked HEL cells in the G2 phase, induced apoptosis, decreased Bcl-2 and caspase-8 protein expression, and increased Bax and caspase-3 expression. In addition, CETSA revealed that PARP1 is an important target gene for the inhibition of HEL cell growth, and SGP-17-S exerted its action on leukemia cells by targeting PARP1. Therefore, this study might provide new solutions and ideas for the treatment of leukemia.

Leveraging a Phage-Encoded Noncanonical Amino Acid: A Novel Pathway to Potent and Selective Epigenetic Reader Protein Inhibitors.

Epigenetic reader proteins interpret histone epigenetic marks to regulate gene expression. Given their vital roles and the link between their dysfunction and various diseases, these proteins present compelling targets for therapeutic interventions. Nevertheless, designing selective inhibitors for these proteins poses significant challenges, primarily due to their unique properties such as shallow binding sites and similarities with homologous proteins. To overcome these challenges, we propose an innovative strategy that uses phage display with a genetically encoded noncanonical amino acid (ncAA) containing an epigenetic mark. This ncAA guides binding to the reader protein's active site, allowing the identification of peptide inhibitors with enhanced affinity and selectivity. In this study, we demonstrate this novel approach's effectiveness by identifying potent inhibitors for the ENL YEATS domain that plays a critical role in leukemogenesis. Our strategy involved genetically incorporating Nε-butyryl-l-lysine (BuK), known for its binding to ENL YEATS, into a phage display library for enriching the pool of potent inhibitors. One resultant hit was further optimized by substituting BuK with other pharmacophores to exploit a unique π-π-π stacking interaction with ENL YEATS. This led to the creation of selective ENL YEATS inhibitors with a KD value of 2.0 nM and a selectivity 28 times higher for ENL YEATS than its close homologue AF9 YEATS. One such inhibitor, tENL-S1f, demonstrated robust cellular target engagement and on-target effects to inhibit leukemia cell growth and suppress the expression of ENL target genes. As a pioneering study, this work opens up extensive avenues for the development of potent and selective peptidyl inhibitors for a broad spectrum of epigenetic reader proteins.

FDA-approved disulfiram as a novel treatment for aggressive leukemia

Acute leukemia continues to be a major cause of death from disease worldwide and current chemotherapeutic agents are associated with significant morbidity in survivors. While better and safer treatments for acute leukemia are urgently needed, standard drug development pipelines are lengthy and drug repurposing therefore provides a promising approach. Our previous evaluation of FDA-approved drugs for their antileukemic activity identified disulfiram, used for the treatment of alcoholism, as a candidate hit compound. This study assessed the biological effects of disulfiram on leukemia cells and evaluated its potential as a treatment strategy. We found that disulfiram inhibits the viability of a diverse panel of acute lymphoblastic and myeloid leukemia cell lines (n = 16) and patient-derived xenograft cells from patients with poor outcome and treatment-resistant disease (n = 15). The drug induced oxidative stress and apoptosis in leukemia cells within hours of treatment and was able to potentiate the effects of daunorubicin, etoposide, topotecan, cytarabine, and mitoxantrone chemotherapy. Upon combining disulfiram with auranofin, a drug approved for the treatment of rheumatoid arthritis that was previously shown to exert antileukemic effects, strong and consistent synergy was observed across a diverse panel of acute leukemia cell lines, the mechanism of which was based on enhanced ROS induction. Acute leukemia cells were more sensitive to the cytotoxic activity of disulfiram than solid cancer cell lines and non-malignant cells. While disulfiram is currently under investigation in clinical trials for solid cancers, this study provides evidence for the potential of disulfiram for acute leukemia treatment.Key messagesDisulfiram induces rapid apoptosis in leukemia cells by boosting oxidative stress.Disulfiram inhibits leukemia cell growth more potently than solid cancer cell growth.Disulfiram can enhance the antileukemic efficacy of chemotherapies.Disulfiram strongly synergises with auranofin in killing acute leukemia cells by ROS induction.We propose testing of disulfiram in clinical trial for patients with acute leukemia.

Encouraging Efficacy Observed in Bexmab Study: A Phase 1/2 Study to Assess Safety and Efficacy of Bexmarilimab in Combination with Standard of Care in Myeloid Malignancies

Background Clever-1 (common lymphatic endothelial and vascular endothelial receptor-1) constitutes a novel macrophage checkpoint. The receptor contributes to immune tolerance by antigen clearing and T cell inhibition. In multiple models, tumor growth is significantly reduced when Clever-1 function is inhibited. Expression of Clever-1 on bone marrow (BM) cells is a prognostic factor for poor outcome in acute myeloid leukemia (AML) and suppression of Clever-1 signaling inhibits leukemia cell growth. Bexmarilimab (BEX), a humanized IgG4 monoclonal antibody, binds Clever-1 resulting in increased antigen presentation, secretion of proinflammatory cytokines and activation of T cells. Clever-1 is highly expressed on leukemic blasts and monocytes of AML and myelodysplastic syndrome (MDS) patients. Treatment of AML bone marrow cells with BEX alone or in combination with azacitidine/venetoclax results in enhanced antigen presentation capacity and increased activation markers on effector T cells with synergistic effect on cell death. Also, BEX treatment alone lowers blast metabolic fitness via interference with mitochondrial oxidation. The ongoing BEXMAB clinical study investigates safety and efficacy of BEX combined with standard of care (SoC) in patients with myeloid malignancies (NCT05428969). Method This is a Ph1/2 study, investigating BEX combined with azacitidine (doublet) in patients with higher risk MDS frontline or hypomethylating agent (HMA)-failed, and relapsed/refractory (r/r) AML patients. BEX is dosed Q1W in 28-day cycles until disease progression or intolerable toxicity. SoC azacitidine is administered as per label. During Ph1, BEX doses were escalated from 1.0 mg/kg to 6.0 mg/kg (Bayesian optimal interval [BOIN] design). BM was analyzed at the end of Cycle 1 and 3 followed by every three cycles. Ph2 will start with a dose optimization per malignancy followed by a dose expansion for efficacy evaluation (2-Stage design). Results Here we report data from the dose finding Ph1 of the doublet. As of 25 July 2023, 22 patients have been enrolled in the doublet (MDS n=5; MDS HMA failed n=5; r/r AML n=12). Baseline characteristics are shown in Table 1. 8/10 MDS/MDS HMA-failed patients were of high to very high risk based on rIPSS; of the r/r AML 7/12 patients had adverse risk based on ELN 2017. The median number of prior therapies for the MDS HMA-failed patients was 1 (range 1-2). The median number of prior therapies for the r/r AML patients was 2 (range 1-4) with all patients having failed prior HMA-containing therapies and 7/12 having received prior HMA plus venetoclax. The majority of observed adverse events (AE) were Grade 1-2 and no dose limiting toxicities have been reported. As of 25 July 2023, a total of 10 BEX related AEs have been reported (8% of all treatment emergent AEs) with four of these events of ≥ Grade 3, two being immune related (capillary leak syndrome and hemophagocytic lymphohistiocytosis). Sustained target engagement of soluble Clever-1 of up to 75% compared to baseline was seen at all tested dose levels in patient blood. Activity of BEX in patient BM was demonstrated using receptor binding assays with BEX treatment resulting in decreased blast Clever-1 expression at all dose levels of the doublet cohort. Increased immune activation as measured using HLA expression and T/NK cell numbers in patient BM was observed after BEX treatment across indications. Up to 2-3-fold increase of CD8 T and NK cells in the BM of BEX treated patients was observed. Preliminary efficacy shows objective responses in 8/15 evaluable patients across all tested BEX doses. Four were observed in patients with prior HMA-failure and four patients stayed on treatment > 6 months. Clinical activity for frontline MDS was seen as one complete remission (CR), marrow CR (mCR) and hematologic improvement for platelets (HI-P); for MDS with HMA failure as one CR, mCR and partial remission (PR); for r/r AML as two incomplete CRs (CRi). Based on accumulating data, responses are observed between end Cycle 1 and end Cycle 4 for all BEX doses. Conclusion Combining BEX plus azacitidine is well-tolerated and shows efficacy across indications. Additional clinical and pharmacodynamic data of the completed Ph1 of the study will be presented during the meeting. Ph2 of BEX plus azacitidine will open in the 2 nd half of 2023 in HMA-failed r/r AML and/or higher risk MDS patients.

HIF1α-mediated transactivation of WTAP promotes AML cell proliferation via m6A-dependent stabilization of KDM4B mRNA.

Hypoxia inducible factor 1α (HIF1α) is abnormally overexpressed in t(8;21) acute myeloid leukemia (AML) and functions as an oncogene through transactivating DNA methyltransferase 3 alpha leading to DNA hypermethylation. However, it remains unclear whether HIF1α influences RNA N6-methyladenosine (m6A) methyltransferases. Here, we show that HIF1α promotes the expression of Wilms tumor 1-associated protein (WTAP), a main component of the m6A methyltransferase complex, markedly alters the transcriptome-wide m6A distribution and enhances cell proliferation in t(8;21) AML. In agreement with this, WTAP is overexpressed and predicts poor prognosis in t(8;21) AML patients. Moreover, WTAP knockdown inhibits growth, and induces apoptosis and differentiation of leukemia cells. Mechanistically, HIF1α transactivates WTAP gene expression by directly binding to the hypoxia-response element of its promoter region. Pharmacological or genetic intervention in the HIF1α-WTAP axis results in the reduction of m6A level on lysine demethylase 4B (KDM4B) transcripts and increased its degradation, correlated with lower expression of KDM4B and higher trimethylation levels of histone H3 on lysine 9. KDM4B knockdown inhibits leukemia cell growth in vitro and in mice. Thus, HIF1α-mediated WTAP high expression enhances the malignant behavior of leukemia cells and drives a crosstalk between m6A RNA methylation and histone methylation through monitoring m6A-dependant KDM4B translation.

Lovastatin inhibits erythroleukemia progression through KLF2-mediated suppression of MAPK/ERK signaling

BackgroundLovastatin, an HMG-CoA inhibitor and an effective cholesterol lowering drug, exhibits anti-neoplastic activity towards several types of cancer, although the underlying mechanism is still not fully understood. Herein, we investigated mechanism of growth inhibition of leukemic cells by lovastatin.MethodsRNAseq analysis was used to explore the effect of lovastatin on gene expression in leukemic cells. An animal model of leukemia was used to test the effect of this statin in vivo. FAM83A and DDIT4 expression was knocked-downed in leukemia cells via lentivirus-shRNA. Western blotting, RT-qPCR, cell cycle analysis and apoptosis assays were used to determine the effect of lovastatin-induced growth suppression in leukemic cells in vitro.ResultsLovastatin treatment strongly inhibited cancer progression in a mouse model of erythroleukemia induced by Friend virus. In tissue culture, lovastatin inhibited cell proliferation through induction of G1 phase cell cycle arrest and apoptosis. Interestingly, lovastatin induced most known genes associated with cholesterol biosynthesis in leukemic cells. Moreover, it suppressed ERK1/2 phosphorylation by downregulating FAM83A and DDIT4, two mediators of MAP-Kinase signaling. RNAseq analysis of lovastatin treated leukemic cells revealed a strong induction of the tumor suppressor gene KLF2. Accordingly, lentivirus-mediated knockdown of KLF2 antagonized leukemia cell suppression induced by lovastatin, associated with higher ERK1/2 phosphorylation compared to control. We further show that KLF2 induction by lovastatin is responsible for lower expression of the FAM83A and DDIT4 oncogenes, involved in the activation of ERK1/2. KLF2 activation by lovastatin also activated a subset of cholesterol biosynthesis genes that may further contribute to leukemia suppression.ConclusionsThese results implicate KLF2-mediated FAM83A/DDIT4/MAPK suppression and activation of cholesterol biosynthesis as the mechanism of leukemia cell growth inhibition by lovastatin.

Trial in Progress: A Phase I/II Study to Assess Safety, Tolerability and Preliminary Efficacy of Bexmarilimab in Combination with Standard of Care in Patients with Hematological Malignancies (BEXMAB)

Background: Emerging data on the role of leukemic cells on shaping the tumor immune environment places macrophages as target cells and immune checkpoints in acute myeloid leukemia (AML) (Miari et. al. Front. Cell Dev. Biol.2021; Sallman et. al. Blood 2019). CLEVER-1 (common lymphatic endothelial and vascular endothelial receptor-1) constitutes such macrophage checkpoint. The receptor contributes to tumor immune tolerance by antigen clearing and T cell inhibition. In multiple models, tumor growth is significantly reduced in CLEVER-1 absence (Viitala et. al. Clin Cancer Res 2019). CLEVER-1 expression level is a prognostic factor for poor outcomes in AML (Pölönen et. al. Cancer Research 2019) and its suppression inhibits leukemia cell growth. Analyses suggests lower expression is associated with venetoclax sensitivity (Lin et al. Mol Ther 2019). Bexmarilimab (FP-1305; BEX), an IgG4 monoclonal antibody, binds CLEVER-1 and alters the function of macrophages and inactive T cells. BEX was shown to increase antigen presentation, induce proinflammatory cytokines and infiltration of activated T cells (Viitala et. al. Clin Cancer Res 2019). The therapeutic potential of BEX is supported with data from the MATINS study (NCT03733990) evaluating single agent in advanced solid tumors. Up to 40% of patients in selected indications experience disease control (stable disease/partial response) associated with a 65% chance of 1-year overall survival (OS) compared to 11% of patients without disease control. BEX treatment is well tolerated without any dose limiting toxicity (DLT) reported. CLEVER-1 is highly expression on malignant blasts and monocytes of bone marrow from AML and myelodysplastic syndrome (MDS) patients. Treatment of these BM cells with BEX alone or in combination with azacitidine or venetoclax results in enhanced antigen presentation capacity and increased activation markers on effector T cells with synergistic effects (EHA 2022 P380). Study Design and Methods: The PhI/II study investigates BEX treatment plus standard of care in MDS, chronic myelomonocytic leukemia (CMML) frontline or hypomethylating agent (HMA)-failed patients or newly diagnosed AML or relapsed/refractory (r/r) AML patients (NCT05428969). Enrollment is ongoing. Ph I is a dose escalation using a Bayesian optimal interval (BOIN) design to explore 4 dose levels of BEX in a cohort size of 5 with 1.0 mg/kg as starting dose. BEX plus azacitidine (doublet) is assessed in a mixed cohort of MDS, CMML frontline and hypomethylating agent (HMA)-failure patients or r/r AML patients. Dose determination may then be performed in separate indications. BEX plus azacitidine + venetocolax (triplet) will follow a similar BOIN design for dose determination in patients with newly diagnosed AML unfit for induction chemotherapy. BEX is dosed in 28-day cycles Q1W during cycles 1-3 followed by Q2W dosing, until disease progression or intolerable toxicity. Standard of care therapy is administered as per label. During Ph II, selected indications are explored for efficacy when BEX is administered at recommended phase 2 dose (RP2D) determined in the doublet and triplet. Dose expansion follows a 2-Stage design with indication-specific target efficacy rates, including complete remission +/- incomplete hematologic recovery, overall response rate or minimal residual disease (MRD)-negativity. Primary endpoints are safety and tolerability, RP2D of BEX and preliminary efficacy. Secondary endpoints include extended efficacy for duration of response, event free survival and OS. Exploratory endpoints are focused on pharmacodynamic parameters for immunological and cellular read-outs and the association of predictive biomarker of BEX clinical activity in different patient sub-groups. Key eligibility includes patient ≥ 18 years of age presenting with morphologically confirmed diagnosis of either MDS (intermediate-very high rIPSS risk), CMML-2 with indication for azacitidine treatment; CMML and MDS patients with failure to HMA therapy, confirmed diagnosis of r/r AML following at least 1 prior treatment line or diagnosis of AML in patients unfit for induction therapy with indication for azacitidine + venetoclax. Patients with APL or myeloproliferative CMML, active auto-immune disorder or requiring systemic corticosteroid or other immunosuppressive treatment are excluded. Approximately 181 patients at 10 US and EU sites will be enrolled.

BCL6 inhibition ameliorates resistance to ruxolitinib in CRLF2-rearranged acute lymphoblastic leukemia.

Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL) is an intractable disease and most cases harbor genetic alterations that activate JAK or ABL signaling. The commonest subtype of Ph-like ALL exhibits a CRLF2 gene rearrangement that brings about JAK1/2-STAT5 pathway activation. However, JAK1/2 inhibition alone is insufficient as a treatment, so combinatorial therapies targeting multiple signals are needed. To better understand the mechanisms underlying the insufficient efficacy of JAK inhibition, we explored gene expression changes upon treatment with a JAK1/2 inhibitor (ruxolitinib) and found that elevated BCL6 expression was one such mechanism. Upregulated BCL6 suppressed the expression of TP53 along with its downstream cell cycle inhibitor p21 (CDKN2A) and pro-apoptotic molecules, such as FAS, TNFRSF10B, BID, BAX, BAK, PUMA, and NOXA, conferring cells some degree of resistance to therapy. BCL6 inhibition (with FX1) alone was able to upregulate TP53 and restore the TP53 expression that ruxolitinib had diminished. In addition, ruxolitinib and FX1 concertedly downregulated MYC. As a result, FX1 treatment alone had growth-inhibitory and apoptosis- sensitizing effects, but the combination of ruxolitinib and FX1 more potently inhibited leukemia cell growth, enhanced apoptosis sensitivity, and prolonged the survival of xenografted mice. These findings provide one mechanism for the insufficiency of JAK inhibition for the treatment of CRLF2-rearranged ALL and indicate BCL6 inhibition as a potentially helpful adjunctive therapy combined with JAK inhibition.

5-Demethylnobiletin Inhibits Cell Proliferation, Downregulates ID1 Expression, Modulates the NF-κB/TNF-α Pathway and Exerts Antileukemic Effects in AML Cells.

Acute myeloid leukemia (AML) is characterized by the dysregulation of hematopoietic cell proliferation, resulting in the accumulation of immature myeloid cells in bone marrow. 5-Demethylnobiletin (5-demethyl NOB), a citrus 5-hydroxylated polymethoxyflavone, has been reported to exhibit various bioactivities, such as antioxidant, anti-inflammatory and anticancer properties. In this study, we investigated the antileukemic effects of 5-demethyl NOB and its underlying molecular mechanisms in human AML cells. We found that 5-demethyl NOB (20–80 μM) significantly reduced human leukemia cell viability, and the following trend of effectiveness was observed: THP-1 ≈ U-937 > HEL > HL-60 > K562 cells. 5-Demethyl NOB (20 and 40 μM) modulated the cell cycle through the regulation of p21, cyclin E1 and cyclin A1 expression and induced S phase arrest. 5-Demethyl NOB also promoted leukemia cell apoptosis and differentiation. Microarray-based transcriptome, Gene Ontology (GO) and Gene Set Enrichment Analysis (GSEA) of differentially expressed genes (DEGs) analysis showed that the expression of inhibitor of differentiation/DNA binding 1 (ID1), a gene associated with the GO biological process (BP) cell population proliferation (GO: 0008283), was most strongly suppressed by 5-demethyl NOB (40 μM) in THP-1 cells. We further demonstrated that 5-demethyl NOB-induced ID1 reduction was associated with the inhibition of leukemia cell growth. Moreover, DEGs involved in the hallmark gene set NF-κB/TNF-α signaling pathway were markedly enriched and downregulated by 5-demethyl NOB. Finally, we demonstrated that 5-demethyl NOB (20 and 40 μM), combined with cytarabine, synergistically reduced THP-1 and U-937 cell viability. Our current findings support that 5-demethyl NOB dramatically suppresses leukemia cell proliferation and may serve as a potential phytochemical for human AML chemotherapy.

Integrated RNAi screening identifies the NEDDylation pathway as a synergistic partner of azacytidine in acute myeloid leukemia

Treatment of acute myeloid leukemia (AML) remains challenging and novel targets and synergistic therapies still need to be discovered. We performed a high-throughput RNAi screen in three different AML cell lines and primary human leukemic blasts to identify genes that synergize with common antileukemic therapies. We used a pooled shRNA library that covered 5043 different genes and combined transfection with exposure to either azacytidine or cytarabine analog to the concept of synthetic lethality. Suppression of the chemokine CXCL12 ranked highly among the candidates of the cytarabine group. Azacytidine in combination with suppression of genes within the neddylation pathway led to synergistic results. NEDD8 and RBX1 inhibition by the small molecule inhibitor pevonedistat inhibited leukemia cell growth. These findings establish an in vitro synergism between NEDD8 inhibition and azacytidine in AML. Taken together, neddylation constitutes a suitable target pathway for azacytidine combination strategies.

Optimization of 4,6-Disubstituted Pyrido[3,2-d]pyrimidines as Dual MNK/PIM Inhibitors to Inhibit Leukemia Cell Growth.

Mitogen-activated protein kinase-interacting kinases (MNKs) and provirus integration in maloney murine leukemia virus kinases (PIMs) are downstream enzymes of cell proliferation signaling pathways associated with the resistance of tyrosine kinase inhibitors. MNKs and PIMs have complementary effects to regulate cap-dependent translation of oncoproteins. Dual inhibitors of MNKs and PIMs have not been developed. We developed a novel 4,6-disubstituted pyrido[3,2-d]pyrimidine compound 21o with selective inhibition of MNKs and PIMs. The IC50's of 21o to inhibit MNK1 and MNK2 are 1 and 7 nM and those to inhibit PIM1, PIM2, and PIM3 are 43, 232, and 774 nM, respectively. 21o inhibits the growth of myeloid leukemia K562 and MOLM-13 cells with GI50's of 2.1 and 1.2 μM, respectively. 21o decreases the levels of p-eIF4E and p-4EBP1, the downstream products of MNKs and PIMs, as well as cap-dependent proteins c-myc, cyclin D1, and Mcl-1. 21o inhibits the growth of MOLM-13 cell xenografts without causing evident toxicity. 21o represents an innovative dual MNK/PIM inhibitor with a good pharmacokinetic profile.

LAMP-5 is an essential inflammatory-signaling regulator and novel immunotherapy target for mixed lineage leukemia-rearranged acute leukemia.

Although great advances have been made in understanding the pathobiology of mixed lineage leukemia-rearranged (MLL-r) leukemias, therapies for this leukemia have remained limited, and clinical outcomes remain bleak. In order to identify novel targets for immunotherapy treatments, we compiled a lineage-independent MLL-r leukemia gene signature using publicly available data sets. Data from large leukemia repositories were filtered through the in silico human surfaceome, providing a list of highly predicted cell surface proteins overexpressed in MLL-r leukemias. LAMP5, a lysosomal associated membrane protein, is expressed highly and specifically in MLL-r leukemia. We found that LAMP5 is a direct target of the oncogenic MLL-fusion protein. LAMP5 depletion significantly inhibited leukemia cell growth in vitro and in vivo. Functional studies showed that LAMP-5 is a novel modulator of innate-immune pathways in MLL-r leukemias. Downregulation of LAMP5 led to inhibition of NF-kB signaling and increased activation of type-1 interferon signaling downstream of Toll-like receptor/interleukin 1 receptor activation. These effects were attributable to the critical role of LAMP-5 in transferring the signal flux from interferon signaling endosomes to pro-inflammatory signaling endosomes. Depletion of IRF7 was able to partially rescue the cell growth inhibition upon LAMP5 downregulation. Lastly, LAMP-5 was readily detected on the surface of MLL-r leukemia cells. Targeting surface LAMP-5 using an antibody-drug conjugate leads to significant cell viability decrease specifically in MLL-r leukemias. Overall, based on the limited expression throughout human tissues, we postulate that LAMP-5 could potentially serve as an immunotherapeutic target with a wide therapeutic window to treat MLL-r leukemias.

Bioactive Potential of Several Actinobacteria Isolated from Microbiologically Barely Explored Desert Habitat, Saudi Arabia.

Simple SummaryBioactive natural products have been regarded as promising tools for treatment of various ailments. Among natural sources, actinomycetes have been widely explored for their potential bioactivity. In this regard, the present study has focused on the phytochemical content and biological activities of several actinobacteria isolates, which were investigated for their phenolic and flavonoid content, as well as their antioxidant, antibacterial and antiprotozoal activities. The most active isolates were further investigated for their antileukemic activity, where such isolates were shown to exert cytotoxic activity against the tested cell lines, following a mechanism that might be due to the ability of the active isolate extracts to reduce cyclooxygenase and lipoxygenase activities. Overall, isolation and characterization of the active molecule from the potential actinomycetes strains will pave the way for the development of drugs against human diseases such as blood cancer.Biomolecules from natural sources, including microbes, have been the basis of treatment of human diseases since the ancient times. Therefore, this study aimed to investigate the potential bioactivity of several actinobacteria isolates form Al-Jouf Desert, Saudi Arabia. Twenty-one actinobacterial isolates were tested for their antioxidant (flavonoids, phenolics, tocopherols and carotenoids) content, and biological activities, namely FRAP, DPPH, ABTS, SOS and XO inhibition, anti-hemolytic and anti-lipid peroxidation as well as their antibacterial and antiprotozoal activities. Accordingly, five isolates (i.e., Act 2, 12, 15, 19 and 21) were selected and their 90% ethanolic extracts were used. The phylogenetic analysis of the 16S rRNA sequences indicated that the most active isolates belong to genus Streptomyces. The genus Streptomyces has been documented as a prolific producer of biologically active secondary metabolites against different cancer types. Thus, the anti-blood cancer activity and the possible molecular mechanisms by which several Streptomyces species extracts inhibited the growth of different leukemia cells, i.e., HL-60, K562 and THP-1, were investigated. In general, the five active isolates showed cytotoxic activity against the tested cell lines in a dose dependent manner. Among the potent isolates, isolate Act 12 significantly decreased the cell viability and showed maximum cytotoxic activities against both HL-60 and K562 cells, while isolate Act 15 exhibited maximum cytotoxic activity against THP-1 cells. Moreover, Act 2 and Act 12 reduced cyclooxygenase (COX-2) and lipoxygenase (LOX) activity, which is involved in the proliferation and differentiation of cancer cells and may represent a possible molecular mechanism underlying leukemia growth inhibition. The bioactive antioxidant extracts of the selected Streptomyces species inhibited leukemia cell growth by reducing the COX-2 and LOX activity. Overall, our study not only introduced a promising natural alternative source for anticancer agents, but it also sheds light on the mechanism underlying the anticancer activity of isolated actinomycetes.

Elucidating the Importance of DOT1L Recruitment in MLL-AF9 Leukemia and Hematopoiesis

Simple SummaryMLL-rearranged leukemia, driven by MLL-fusion proteins, is an aggressive, therapy-resistant leukemia found in >60% of infant leukemia and ~10% of adult leukemia. Studies have shown that inhibiting DOT1L enzymatic activity blocks leukemogenesis. However, DOT1L is critical for various normal cellular functions, including hematopoiesis. This study aimed to show that targeting the interaction between the MLL-AF9 fusion and DOT1L would inhibit leukemogenesis while sparing non-leukemic hematopoiesis. We found that disrupting the AF9-DOT1L interaction with a single point mutation was sufficient to impair leukemogenesis. We also demonstrate that genetic interventions that result in loss of DOT1L enzymatic activity in non-leukemic cells rapidly depletes hematopoietic stem and progenitor cells within 7–10 days; however, hematopoiesis was preserved when the AF9-DOT1L interaction was disrupted, leaving the enzymatic function intact. These studies are a proof of concept demonstrating the potential therapeutic advantage of inhibiting the AF9-DOT1L interaction and disrupting the integrity of the MLL-fusion complex.MLL1 (KMT2a) gene rearrangements underlie the pathogenesis of aggressive MLL-driven acute leukemia. AF9, one of the most common MLL-fusion partners, recruits the histone H3K79 methyltransferase DOT1L to MLL target genes, constitutively activating transcription of pro-leukemic targets. DOT1L has emerged as a therapeutic target in patients with MLL-driven leukemia. However, global DOT1L enzymatic inhibition may lead to off-target toxicities in non-leukemic cells that could decrease the therapeutic index of DOT1L inhibitors. To bypass this problem, we developed a novel approach targeting specific protein-protein interactions (PPIs) that mediate DOT1L recruitment to MLL target genes, and compared the effects of enzymatic and PPIs inhibition on leukemic and non-leukemic hematopoiesis. MLL-AF9 cell lines were engineered to carry mutant DOT1L constructs with a defective AF9 interaction site or lacking enzymatic activity. In cell lines expressing a DOT1L mutant with defective AF9 binding, we observed complete disruption of DOT1L recruitment to critical target genes and inhibition of leukemic cell growth. To evaluate the overall impact of DOT1L loss in non-leukemic hematopoiesis, we first assessed the impact of acute Dot1l inactivation in adult mouse bone marrow. We observed a rapid reduction in myeloid progenitor cell numbers within 7 days, followed by a loss of long-term hematopoietic stem cells. Furthermore, WT and PPI-deficient DOT1L mutants but not an enzymatically inactive DOT1L mutant were able to rescue sustained hematopoiesis. These data show that the AF9-DOT1L interaction is dispensable in non-leukemic hematopoiesis. Our findings support targeting of the MLL-AF9–DOT1L interaction as a promising therapeutic strategy that is selectively toxic to MLL-driven leukemic cells.

Identification of the novel Np17 oncogene in human leukemia.

We previously defined the HERV-K Np9 as a viral oncogene. Here we report the discovery of a novel oncogene, Np17, which is homologous to the viral Np9 gene and predominantly present in Hominoidea. Np17 is located on chromosome 8, consists of 7 exons, and encodes a 16.8kDa nuclear protein with149 amino-acid residue. Functionally, knockdown of Np17 induced growth inhibition of leukemia cells, whereas enforced expression of Np17 promoted growth of leukemia cells in vitro and in vivo. In human leukemia, Np17 was detected in 59.65% (34/57) of acute myeloid leukemia (AML) patients examined and associated with refractory/relapsed AML. Mechanistically, Np17 decreased p53 levels and its mechanism might be involved in recruiting nuclear MDM2 to p53 for ubiquitin-mediated degradation. These findings reveal that Np17 is a novel oncogene associated with refractory/relapsed leukemia.

Enhanced anticancer activity and endocytic mechanisms by polymeric nanocarriers of n-butylidenephthalide in leukemia cells

The purpose of this study was to investigate the antitumor mechanisms of n-butylidenephthalide (BP) and to further examine the delivery efficacy of polycationic liposome containing PEI and polyethylene glycol complex (LPPC)-encapsulated BP in leukemia cells. MTS, flow cytometric and TUNEL assays were performed to assess cell viability and apoptosis. BP and BP/LPPC complex delivery efficiency was analyzed by full-wavelength fluorescent scanner and fluorescence microscope. The expressions of cell cycle- and apoptosis-related proteins were conducted by Western blotting. The results showed that BP inhibited leukemia cell growth by inducing cell cycle arrest and cell apoptosis. LPPC-encapsulated BP rapidly induced endocytic pathway activation, resulting in the internalization of BP into leukemia cells, causing cell apoptosis within 1h. LPPC encapsulation enhanced the cytotoxic activity of BP and did not influence the effects of BP induction that suggested LPPC-encapsulated BP might be developed as anti-leukemia drugs in future.

Discovery of M-808 as a Highly Potent, Covalent, Small-Molecule Inhibitor of the Menin-MLL Interaction with Strong In Vivo Antitumor Activity.

Targeting the menin-MLL protein-protein interaction is a new therapeutic strategy for the treatment of acute leukemia carrying MLL fusion (MLL leukemia). We describe herein the structure-based optimization of a class of covalent menin inhibitors, which led to the discovery of M-808 (16) as a highly potent and efficacious covalent menin inhibitor. M-808 effectively inhibits leukemia cell growth at low nanomolar concentrations and is capable of achieving partial tumor regression in an MV4;11 xenograft tumor model in mice at a well-tolerated dose schedule. Determination of the co-crystal structure of M-808 in complex with menin provides a structural basis for their high-affinity, covalent interactions. M-808 represents a promising, covalent menin inhibitor for further optimization and evaluation toward developing a new therapy for the treatment of MLL leukemia.

Jujuboside B promotes the death of acute leukemia cell in a RIPK1/RIPK3/MLKL pathway-dependent manner

Initiation of necroptosis has been considered as a promising strategy for anticancer therapies, especially for eradicating apoptosis-resistant malignant cells. Jujubisode B is a natural saponins extracted from the seeds of Zizyphi Spinosi Semen, and possesses multiple pharmacological activities, including antianxiety, anti-inflammation, antiplatelet aggregation and induction of apoptosis. This study aims to explore the effect of jujuboside B on acute leukemic cells and the underlying mechanisms. Our results showed that jujuboside B inhibited leukemia cell growth in a dose-dependent manner and attenuated the clonogenic ability of U937 cells, concomitant with activation of RIPK1/RIPK3/MLKL pathway; these phenomena were evidently blocked by necroptosis inhibitor (Nec-1). With the help of Molecular Operating Environment (MOE) program, we identified that RIPK1, RIPK3 and MLKL are potential targets of jujuboside B. To the best of our knowledge, this is the first study to provide evidence that jujuboside B possesses antileukemic activity via a mechanism involving activation of RIPK1/RIPK3/MLKL pathway.

PF245 METFORMIN INHIBITS THE GROWTH OF MYELOID LEUKEMIA CELLS PARTLY DUE TO SUPPRESSION OF TYRO3 EXPRESSION

Background:Metformin, an anti‐diabetic drug, is known to inhibit leukemia cell growth mainly through activation of AMP‐activated protein kinase (AMPK), resulting in inhibition of mTORC1 signaling. Meanwhile, TYRO3 is a member of the TAM family (TYRO3, AXL, and MERTK) of receptor tyrosine kinases. While the roles of activated AXL and MERTK in leukemia cell growth have been reported by us and others, the roles of TYRO3 have not been well elucidated.Aims:We examined the involvement of TYRO3 in anti‐leukemia effects of metformin.Methods:Four myeloid leukemia cell lines (OCI‐AML2, OCI‐AML3, AA, and K562) expressing TYRO3 protein were used. AA is a human cell line derived from pure erythroid leukemia. All cell lines were cultured with increasing concentrations of metformin. To knockdown TYRO3 expression, small interfering RNA (siRNA) against TYRO3 or control siRNA was transfected using an electroporation system. Cell growth was assessed with a colorimetric assay after culturing for three days. Expression levels and the activation of various signaling proteins in lysates from cultured cells were examined using immunoblotting. Expression levels of mRNA were examined by quantitative RT‐PCR.Results:Treatment with metformin suppressed the growth of these four cell lines in a dose‐dependent manner. Immunoblotting revealed that metformin increased phosphorylated AMPK while decreased phosphorylation of mTOR and the downstream proteins such as 4E‐BP1 and P70S6K without affecting expression levels of those proteins in general. Moreover, metformin treatment decreased expression levels of TYRO3 and c‐MYC and phosphorylation of TYRO3, STAT3, and ERK1/2. In K562 cells treated with metformin, decrease in phosphorylated TYRO3 was more evident than decrease in TYRO3 expression levels. In OCI‐AML2, OCI‐AML3, and K562 cells, which express AXL, metformin treatment also decreased expression levels and phosphorylation of AXL. Quantitative RT‐PCR analysis showed that expression of TYRO3 and MYC mRNA decreased by metformin treatment. Next, siRNA experiments showed that TYRO3 knockdown suppressed the cell growth to approximately 50% of the control. It also suppressed phosphorylation of STAT3 and ERK1/2, which are downstream molecules of TYRO3 signaling, while it did not obviously suppress the phosphorylation of mTOR.Summary/Conclusion:We revealed that metformin treatment suppressed TYRO3 expression in myeloid leukemia cells for the first time. We also found that TYRO3 is involved in leukemia cell growth because TYRO3 knockdown suppressed the growth. From these findings, we showed that growth inhibition by metformin might be partly due to its suppression of TYRO3. In leukemia cells expressing AXL, suppression of AXL expression might also be involved in growth inhibition by metformin. It is widely speculated that growth inhibition by metformin is mainly due to inhibition of mTORC1 signaling through AMPK activation and some AMPK‐independent mechanisms. We showed here that suppression of TYRO3 and/or AXL by metformin might also be involved in growth inhibition, although the individual effects of AMPK‐mTORC1 and TYRO3/AXL pathways are not clearly distinguished because mTOR is one of the downstream molecules of TYRO3/AXL pathway. In clinical settings in the future, metformin treatment might be effective for patients with leukemia cells expressing TYRO3 and/or AXL proteins as well as mTOR. Detection of expression and activation of these proteins can be done to select the appropriate patients for administration of metformin.image

CDK8 Inhibitor SEL120-34A Has Therapeutic Efficacy in Murine and Human Acute Myeloid Leukemia Models

MJ and JF provided equal contribution as senior authorsAcute myeloid leukemia (AML) is associated with poor survival and characterized by an accumulation of immature myeloid blasts in the bone marrow. To efficiently target AML, new therapies directed to leukemia stem cells (LSCs), a self-renewing population that constitutes a chemo-resistant reservoir responsible for disease relapse, are warranted.Cyclin-dependent kinase 8 inhibitors' (CDK8i) anti-cancer activity has been demonstrated in human acute myeloid leukemia (AML) cell lines. Efficacy has been associated with activation of super enhancer regions and reduction of STAT5 S726 phosphorylation in sensitive cells (Pelish, Nature, 2015). SEL120-34A (Selvita, Poland), a selective low nanomolar CDK8/CDK19 inhibitor, has been shown to have antileukemic effect in a panel of AML cell lines (Rzymski, Oncotarget 2017).To evaluate whether primitive AML cells, enriched for LSCs, are sensitive to CDK8 inhibition, we tested the CDK8i SEL120-34A and Senexin B on TEX cells, an AML cell line that exhibits a hierarchical organization and can be used as a LSC model (Warner, Leukemia, 2005). Treatment of TEX cells with SEL120-34A or Senexin B resulted in an inhibition of cell growth (IC50 of 8 and 31 nM respectively at 10 days of culture), associated with reduced activation of STAT5 and STAT1. Both STAT proteins were previously identified as biomarkers for SEL120-34A activity (Rzymski, Oncotarget 2017). In addition, RNA sequencing followed by gene-set enrichment analysis (GSEA) revealed a loss of a LSC signature.To functionally address the effects of CDK8i on LSCs, we used a murine dsRed+ AML model driven by retroviral MLL-AF9 expression. This model has a well-defined LSC population and initiates AML with a short latency, enabling rapid follow-up experiments in syngeneic hosts. Treatment of c-Kit+ AML cells with SEL120-34A or Senexin B in vitro resulted in strong inhibition of leukemia cell growth (IC50 of 119 and 143 nM, respectively, at 7 days of culture), associated with increased apoptosis and reduced cycling of the cells. To address the in vivo therapeutic efficacy of SEL120-34A, mice injected with AML cells 10 days earlier were treated orally for 12 consecutive days using doses of 20 and 40 mg/kg before sacrificed. No tolerability issues were observed with mice maintaining a stable weight through the treatment. Whereas the control group had 87% leukemia cells in the peripheral blood at the end-point analysis, SEL120-34A treated animals showed a dose-dependent selective anti-leukemic activity with a corresponding 78% (20 mg/kg) and 67% (40 mg/kg, p=0.011) of leukemia cells. Similarly, a significant selective dose-dependent anti-leukemic activity of SEL120-34A was observed also in the bone marrow. In addition, a dose-dependent reduced white blood cell count and smaller spleen size upon SEL120-34A treatment was observed, demonstrating that CDK8 inhibition has selective anti-leukemic activity in vivo. Notably, SEL120-34A treatment resulted in granulocytic (Gr1+CD11b-) differentiation of the AML cells (5.8% of the AML cells in the control group; 21.9% at 20 mg/kg, p=0.03; and 22.3%, p=0.0037 at 40 mg/kg). Moreover, SEL120-34A treatment resulted in strong inhibition of Stat5 S726 and Stat1 S727 phosphorylation in AML bone marrow cells harvested from the mice.To test the efficacy of CDK8 inhibition on AML patient cells, four AML patient derived xenografts were treated with SEL120-34A ex vivo. In all four samples, two of which contained activating mutations in FLT3, SEL120-34A treatment resulted in a significant antileukemic activity with decreased number of AML cells and an increase in apoptotic cells.Taken together, our data from murine and human AML models indicate that CDK8 inhibition has therapeutic efficacy in primitive AML cells. SEL120-34A treatment in vivo resulted in reduced leukemia cell burden in both blood and bone marrow accompanied by granulocytic differentiation. Treatment of AML cells in cultures consistently resulted in a reduction in AML cell number and increased apoptosis. Ongoing and future experiments will address whether SEL120-34A treatment also extends survival of mice with AML in syngeneic and patient-derived xenograft models. These data highlight CDK8 as a promising therapeutic target in AML and provides preclinical proof of concept for anti-leukemic efficacy of the clinical candidate SEL120-34A in relevant AML models. DisclosuresMazan:Selvita S.A.: Employment. Majewska:Selvita S.A.: Employment. Wiklik:Selvita S.A.: Employment. Combik:Selvita S.A.: Employment. Masiejczyk:Selvita S.A.: Employment. Fiedor:Selvita S.A.: Employment. Obacz:Selvita S.A.: Employment. Bialas:Selvita S.A.: Employment. Chesy:Selvita S.A.: Employment. Gabor-Worwa:Selvita S.A.: Employment. Brzózka:Selvita S.A.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Rzymski:Selvita S.A.: Employment, Equity Ownership. Fioretos:Cantargia: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.