Resistant Acute Myeloid Leukemia Cell Research Articles

All-Trans Retinoic Acid Enhances the Anti-Leukemia Effect of Venetoclax on Acute Myeloid Leukemia Cells

Objective: Acute myeloid leukemia (AML) is a malignant disease of the hematopoietic system in which arrested maturation, excessive proliferation and inhibition of apoptosis have been observed in the process of hematopoietic stem cell differentiation. Some patients, especially elderly patients, do not tolerate cytotoxic chemotherapy, and some patients are resistant to chemotherapeutic drugs, consequently molecular targeting therapy has brought new hope for AML treatment. Venetoclax (ABT-199) is a selective inhibitor of the antiapoptotic protein Bcl-2. Increased expression of Bcl-2 is an important factor in the inhibition of apoptosis in leukemia cells. The use of a single drug is often limited because of resistance, and a better curative effect can be obtained in combination with other drugs. All-trans retinoic acid (all-trans-retinoic acid, ATRA) was first used by Chinese doctor to treat acute promyelocytic leukemia (APL). Many studies have shown that ATRA can inhibit the expression of Bcl-2 in AML cells and promote apoptosis. We aimed to test for the first time whether the combination of ATRA and venetoclax would produce a stronger anti-AML effect. Methods: The effects of ATRA and ABT-199 on the ABT-199-resistant AML cell lines OCI-AML3 and THP-1 and the sensitive cell lines MV-4-11 and MOLM-13 were observed in vitro. The following methods were used in this study. 1. CCK8 assay was used to detect cell proliferation. 2. Annexin V/FITC and PI double staining were used to detect apoptosis rate by flow cytometry. 3. Flow cytometry was used to detect the expression of CD11b. 4. PI single staining and flow cytometry were used to study the cell cycle. 5. Western blotting was used to detect the expression of Bcl-2, Bax, Mcl-1, PARP, caspase3, GSK-3β and Cyclin-D1 proteins in OCI-AML3 cells. Results:1. Compared with the control group and the ATRA or ABT-199 single drug group, cell proliferation was significantly inhibited in the combined drug group, the apoptosis rate was significantly increased, and the cell cycle was blocked in G1 phase. 2. The expression of CD11b in the ATRA or ABT-199 group and combination group was significantly higher than that in the control group. 3. In OCI-AML3 cells, compared with the ABT-199 drug group, the expression of Bcl-2, Mcl-1 and Cyclin-D1 decreased, and the expression of Bax, cleaved-PARP, cleaved caspase3 and p-GSK-3β expression increased in the combined treatment group. Conclusion: All-trans retinoic acid (ATRA) can enhance the anti-leukemia effect of ABT-199 on AML cell lines by inhibiting cell proliferation as seen by the cell cycle arrest of AML cells in G1 phase, promoting apoptosis and inducing differentiation. ATRA can reverse the drug resistance of AML cells to ABT-199 by inhibiting the expression of Bcl-2 and Mcl-1. Our observations may provide new strategies and theoretical support for the clinical treatment of AML. Key Wor ds :AML, Venetoclax, ATRA, Apoptosis Disclosures No relevant conflicts of interest to declare.

HIF-1α forms regulatory loop with YAP to coordinate hypoxia-induced adriamycin resistance in acute myeloid leukemia cells.

Despite the improvement in acute myeloid leukemia (AML) treatments, most patients had a poor prognosis and suffered from chemoresistance and disease relapse. Therefore, there is an urgent need for elucidation of mechanism(s) underlying drug resistance in AML. In the present study, we found that AML cells showed less susceptibility to adriamycin (ADR) in the presence of hypoxia, while inhibition of hypoxia-inducible factor 1α (HIF-1α) by CdCl2 can make AML cells re-susceptibile to ADR even under hypoxia. Moreover, HIF-1α is overexpressed and plays an important role in ADR-resistance maintenance in resistant AML cells. We further found hypoxia or induction of HIF-1α can significantly upregulate yes-associated protein (YAP) expression in AML cells, and resistant cells express a high level of YAP. Finally, we found that YAP may not only enhance HIF-1α stability but also promote HIF-1α's activity on the target gene pyruvate kinase M2. In conclusion, our data indicate that HIF-1α or YAP may represent a therapeutic target for overcoming resistance toward adriamycin-based chemotherapy in AML.

Overcoming adaptive therapy resistance in AML by targeting immune response pathways.

Targeted inhibitors to oncogenic kinases demonstrate encouraging clinical responses early in the treatment course; however, most patients will relapse because of target-dependent mechanisms that mitigate enzyme-inhibitor binding or through target-independent mechanisms, such as alternate activation of survival and proliferation pathways, known as adaptive resistance. Here, we describe mechanisms of adaptive resistance in FMS-like receptor tyrosine kinase (FLT3)-mutant acute myeloid leukemia (AML) by examining integrative in-cell kinase and gene regulatory network responses after oncogenic signaling blockade by FLT3 inhibitors (FLT3i). We identified activation of innate immune stress response pathways after treatment of FLT3-mutant AML cells with FLT3i and showed that innate immune pathway activation via the interleukin-1 receptor-associated kinase 1 and 4 (IRAK1/4) complex contributes to adaptive resistance in FLT3-mutant AML cells. To overcome this adaptive resistance mechanism, we developed a small molecule that simultaneously inhibits FLT3 and IRAK1/4 kinases. The multikinase FLT3-IRAK1/4 inhibitor eliminated adaptively resistant FLT3-mutant AML cells in vitro and in vivo and displayed superior efficacy as compared to current targeted FLT3 therapies. These findings uncover a polypharmacologic strategy for overcoming adaptive resistance to therapy in AML by targeting immune stress response pathways.

CircPAN3 contributes to drug resistance in acute myeloid leukemia through regulation of autophagy

The aim of this study was to investigate the role and underlying mechanism of circular RNA (circRNA) circPAN3 in mediating drug resistance in acute myeloid leukemia (AML). We first established two doxorubicin (ADM)-resistant AML cell lines and then utilized high-throughput RNA sequencing (RNA-seq) to compare their circRNA expression profiles with those of the parental cell lines. With bioinformatic analysis, we identified key circRNA molecules involved in drug resistance and validated our findings in clinical specimens. The target microRNAs (miRNAs) and downstream mRNAs were also explored bioinformatically. Using RNA interference technique, the potential mechanism was further investigated. Twenty-nine circRNAs were identified to be differentially expressed between ADM-resistant and sensitive cells. We found that circPAN3 is most likely a key mediator in the development of AML drug resistance, evidenced by the increased expression in ADM-resistant cell lines and BM samples from relapsed patients. Additionally, downregulation of circPAN3 by small interfering RNA (siRNA) significantly restored drug sensitivity to ADM in the two ADM-resistant cell lines, but lentivirus-mediated circPAN3 overexpression had the opposite effect. Subsequent bioinformatic analysis and mechanistic experiments revealed that circPAN3 may facilitate AML drug resistance through regulating autophagy and influencing expression of apoptosis-related proteins, while AMPK/mTOR signaling plays a key role in the regulation of circPAN3 on autophagy. These findings may provide new important insights into the role of circRNAs in mediating AML drug resistance, and suggest that circPAN3 might be a potential target for treatment of drug-resistance AML, which merits further investigation and validation.

Abstract 297: Activity of carfilzomib (CFZ) alone and in combination with cytarabine and chloroquine diphosphate (CHQ) in acute myeloid leukemia (AML)

Abstract Background: Despite the recent advances in AML with the emergence of multiple targeted therapies, patient outcomes remain poor, particularly in the relapsed/refractory setting underscoring the need for novel therapeutic strategies. Proteasome inhibitors (PIs) have antitumor activity in AML through inhibition of the nuclear factor κB pathway and induction of apoptosis with multiple clinical trials showing promising activity of bortezomib particularly when combined with other cytotoxic agents (Csizmar 2016). CFZ, a second-generation PI, has preferential preclinical activity in AML compared to bortezomib and is therefore an agent of interest in AML therapy (van der Helm 2015). Here we assessed the activity of CFZ alone and in combination with cytarabine and the autophagy inhibitor CHQ in AML cell lines. Methods: After screening 18 AML cell lines, 3 sensitive: KASUMI1, ML2, and MV411, and 3 resistant cell lines: AML193, THP1, and NOMO1, were selected for further analysis using an IC50 cutoff for CFZ of 10 nM. Apoptosis, cell cycle, and cell senescence analysis were performed after 72 hours of CFZ exposure at 10 nM. Combination assays using CFZ 10 nM, cytarabine 200 nM, and CHQ 20 µM were performed to evaluate for potential interaction in the form of antagonism or potentiation. Proteomic analysis was performed at baseline using reverse phase protein assay (RPPA) and confirmed with western blot analysis. Results: Single-agent CFZ induced apoptosis in sensitive cell lines with apoptotic rates increasing by 85% to 95%; while apoptotic rates remained unchanged in resistant cell lines. Similarly, CFZ resulted in G0/G1 cell cycle arrest in sensitive but not resistant AML cell lines. Lack of difference in cellular senescence confirmed apoptosis and cell cycle arrest as the major mechanisms of CFZ-induced growth inhibition in AML cell lines. No antagonism was noted when CFZ 10 nM was combined with cytarabine (200 nM). RPPA revealed that AML cell lines with low expression of autophagy related proteins are more sensitive to proteasome inhibition with CFZ treatment compared to resistant cell lines. This was confirmed with western blot which showed upregulation of the autophagy related proteins in resistant cell lines upon exposure to CFZ. However, sensitive cell lines did not upregulate autophagy pathways which may lead to cell cycle arrest and apoptosis. Inhibiting autophagy with CHQ sensitized CFZ resistant lines to CFZ treatment. Conclusion: CFZ demonstrated single agent activity in human AML cell lines and upregulation of autophagy may contribute to resistance to CFZ which can be overcome using autophagy inhibitors such as CHQ. These results support the development of CFZ and autophagy inhibitor-based combinations for AML. Citation Format: Mao Yu Peng, Yasmin Abaza, Dylan Conklin, Erika Von Euw, Monica Mead, John Timmerman, Dennis Slamon, Sarah Larson. Activity of carfilzomib (CFZ) alone and in combination with cytarabine and chloroquine diphosphate (CHQ) in acute myeloid leukemia (AML) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 297.

Current Therapeutic Results and Treatment Options for Older Patients with Relapsed Acute Myeloid Leukemia.

Considerable progress has been made in the treatment of acute myeloid leukemia (AML). However, current therapeutic results are still unsatisfactory in untreated high-risk patients and poorer in those with primary refractory or relapsed disease. In older patients, reluctance by clinicians to treat unfit patients, higher AML cell resistance related to more frequent adverse karyotype and/or precedent myelodysplastic syndrome, and preferential involvement of chemorefractory early hemopoietic precursors in the pathogenesis of the disease further account for poor prognosis, with median survival lower than six months. A general agreement exists concerning the administration of aggressive salvage therapy in young adults followed by allogeneic stem cell transplantation; on the contrary, different therapeutic approaches varying in intensity, from conventional salvage chemotherapy based on intermediate–high-dose cytarabine to best supportive care, are currently considered in the relapsed, older AML patient population. Either patients’ characteristics or physicians’ attitudes count toward the process of clinical decision making. In addition, several new drugs with clinical activity described as “promising” in uncontrolled single-arm studies failed to improve long-term outcomes when tested in larger randomized clinical trials. Recently, new agents have been approved and are expected to consistently improve the clinical outcome for selected genomic subgroups, and research is in progress in other molecular settings. While relapsed AML remains a tremendous challenge to both patients and clinicians, knowledge of the molecular pathogenesis of the disease is fast in progress, potentially leading to personalized therapy in most patients.

MiR-9 Enhances the Chemosensitivity of AML Cells to Daunorubicin by Targeting the EIF5A2/MCL-1 Axis

Daunorubicin (Dnr) is at the forefront of acute myeloid leukemia (AML) therapy, but drug resistance poses a major threat to treatment success. MicroRNA (miR)-9 has been shown to have a pivotal role in AML development. However, little is known about the role of miR-9 in Dnr resistance in AML. We explored the potential role of miR-9 in Dnr resistance in AML cells and its mechanism of action. AML cell lines with high half-maximal inhibitory concentration to Dnr in vivo had significantly low miR-9 expression. miR-9 overexpresssion sensitized AML cells to Dnr, inhibited cell proliferation, and enhanced the ability of Dnr to induce apoptosis; miR-9 knockdown had the opposite effects. Mechanistic studies demonstrated that eukaryotic translation initiation factor 5A-2 (EIF5A2) was a putative target of miR-9, which was inversely correlated with the expression and role of miR-9 in AML cells. miR-9 improved the anti-tumor effects of Dnr by inhibiting myeloid cell leukemia-1 (MCL-1) expression, which was dependent on downregulation of EIF5A2 expression. These results suggest that miR-9 has an essential role in Dnr resistance in AML cells through inhibition of the EIF5A2/MCL-1 axis in AML cells. Our data highlight the potential application of miR-9 in chemotherapy for AML patients.

TUG1 knockdown enhances adriamycin cytotoxicity by inhibiting glycolysis in adriamycin-resistant acute myeloid leukemia HL60/ADR cells.

Taurine-upregulated gene 1 (TUG1) has been reported as an oncogenic long non-coding RNA (lncRNA) in acute myeloid leukemia (AML). Nevertheless, the roles and molecular mechanism of TUG1 in drug resistance of AML cells are still unclear. Glycolysis level was evaluated by detecting glucose consumption and lactate production. qRT-PCR and Western blot were performed to detect TUG1, hexokinase2 (HK2) and pyruvate kinase isoenzyme M2 (PKM2) expressions. Adriamycin (ADR) cytotoxicity and apoptosis were assessed by MTT assay and flow cytometry, respectively. The changes of the protein kinase B (Akt) pathway were determined by Western blot analysis of phosphorylated-Akt (p-Akt) (ser473) and Akt. Our results showed that glycolysis was increased in HL60/ADR cells, as evidenced by the elevated glucose consumption and lactate production, as well as the increased HK2 and PKM2 expressions at mRNA and protein levels. TUG1 was up-regulated in HL60/ADR cells and TUG knockdown inhibited glycolysis. TUG1 knockdown enhanced ADR-induced cytotoxicity and apoptosis in HL60/ADR cells. TUG1 knockdown inhibited the Akt pathway and activation of the Akt pathway by 740Y-P attenuated the effects of TUG1 knockdown on ADR-induced cytotoxicity and apoptosis, as well as glycolysis in HL60/ADR cells. Taken together, TUG1 knockdown enhances adriamycin cytotoxicity in HL60/ADR cells via inhibiting the glycolysis by inactivating the Akt pathway.

Synergistic AML Cell Death Induction by Marine Cytotoxin (+)-1(R), 6(S), 1'(R), 6'(S), 11(R), 17(S)-Fistularin-3 and Bcl-2 Inhibitor Venetoclax.

Treatment of acute myeloid leukemia (AML) patients is still hindered by resistance and relapse, resulting in an overall poor survival rate. Recently, combining specific B-cell lymphoma (Bcl)-2 inhibitors with compounds downregulating myeloid cell leukemia (Mcl)-1 has been proposed as a new effective strategy to eradicate resistant AML cells. We show here that 1(R), 6(S), 1’(R), 6’(S), 11(R), 17(S)-fistularin-3, a bromotyrosine compound of the fistularin family, isolated from the marine sponge Suberea clavata, synergizes with Bcl-2 inhibitor ABT-199 to efficiently kill Mcl-1/Bcl-2-positive AML cell lines, associated with Mcl-1 downregulation and endoplasmic reticulum stress induction. The absolute configuration of carbons 11 and 17 of the fistularin-3 stereoisomer was fully resolved in this study for the first time, showing that the fistularin we isolated from the marine sponge Subarea clavata is in fact the (+)-11(R), 17(S)-fistularin-3 stereoisomer keeping the known configuration 1(R), 6(S), 1’(R), and 6’(S) for the verongidoic acid part. Docking studies and in vitro assays confirm the potential of this family of molecules to inhibit DNA methyltransferase 1 activity.

Global Gene Expression Analysis Reveals Genotoxic Effect of Nrf2 Pharmacological Inhibitor Brusatol in Myeloid Leukemia Cells- a Promising Novel Anticancer Agent

Combating drug resistance is a paramount task in treating acute myeloid leukemia (AML). NFE2 related factor 2 (NRF2) is a major player in inducing chemo resistance in both AML and solid tumors. We have previously reported that inhibition of NRF2 both by shRNA knockdown and pharmacological inhibition using brusatol sensitizes inherent resistant AML cell lines and patient samples to chemotherapy (Karathedath et al, Plos one, 2017). Recent studies have shown that brusatol acts as a potent protein translation inhibitor in A549 lung cancer cell line and this deleterious effect is due to the molar scale concentrations of the drug (Stokoe, et al., 2016, Harder et al, 2016).As the available reports was in solid tumor lines and at higher concentrations, we undertook this study to elucidate the mechanism of action of brusatol in our experimental setup utilizing Nano molar concentration of brusatol in AML cell lines. We compared the global gene expression changes induced by brusatol in AML cell lines resistant to Ara-C, Dnr and ATO namely THP-1 and U937.Total RNA was extracted from both cell lines with and without brusatol treatment (100nM for 6 hours) using Qiagen RNeasy mini kit, cDNA was synthesized and subjected to a One -color 8X60K Agilent micro-array analysis. Data normalization, and analysis was performed using Gene Spring GX (V 12.0) software using the 75th percentile shift. Using student t-Test, 3070 genes were identified to be differentially expressed between THP-1 control and brusatol treated while 6856 genes were identified to be differentially expressed between U937 control and brusatol treated samples. About 1242 differentially expressed genes were common between brusatol treated THP-1 and U937. Differentially expressed genes(DEGs) were clustered using hierarchical clustering based on Pearson coefficient correlation algorithm to identify significant gene expression patterns. Genes were classified based on functional category using biological analysis tool DAVID. The enriched genes in both THP-1 and U937 after brusatol treatment fell into the following; Molecular function: protein binding (1396 and 1701 genes), Biological function: regulation of transcription (350 and 502 genes), Cellular component: Mitochondrion (231 and 249 genes) and so on. KEGG analysis was performed to identify enriched pathways after brusatol treatment in DEGs. DEGs in THP1 and U937 were enriched in 33 and 57 pathways were respectively, combining the pathways revealed 20 common target pathways between THP1 and U937. Pathways involved in cellular stress response such as NFkB, TNF and MAPK pathways were selectively up-regulated in the common target pathways and genes involved in cell cycle pathway were down regulated. Select genes from these target pathways and few genes DOCK3, KLF10, NR4A3, GADD45B, KDM4B, GPR155, H2FAY, from the top 100 DEGs were validated by RQ-PCR (SyBr green) all the genes screened showed results consistent with the micro-array data.Interestingly DNA damage response genes GADD45B, KDM4B, H2AFY were in the top up-regulated genes indicating that brusatol might also cause DNA damage in leukemic cells. NR4A3 the orphan nuclear receptor is an established tumor suppressor in AML and is generally silenced in both AML cell lines and primary samples. One of the key finding from our data is that brusatol strikingly up-regulated NRA4A3 expression in both THP1 and U937. This study has shed light into other modes of action of brusatol apart from being a modulator of the protein translational machinery. Mechanism of chemo sensitization by brusatol could be tissue, time and concentration specific. Our data reveals brusatol at nano-molar concentration acts as a potent DNA damaging agent and reactivates NR4A3 expression in AML apart from being an inhibitor of NRF2. We have also reported at nano-molar concentration brusatol has minimal toxicity towards normal Peripheral blood mono-nuclear cells. Brusatol could be an ideal compound to combat drug resistance in AML. Further experimental validation is warranted to substantiate these finding of the mode of action of brusatol in chemo sensitizing AML cells. DisclosuresNo relevant conflicts of interest to declare.

Integrative Analysis of AML Cell Response to Cytarabine Reveals Synergistic Opportunities Centered on Cholesterol Metabolism

Background: Despite spectacular advances in deciphering the molecular landscape of acute myeloid leukemia (AML), the clinical course of this disease has not improved dramatically for decades. Resistance to therapy and disease recurrence almost invariably occur, highlighting the need for a deeper understanding of these processes. Our general strategy takes into account decreased oxygen (O2) tension, a micro-environmental hallmark which remains understudied in the context of AML. While low O2 compartments such as bone marrow niches are well-recognized hosts of drug resistant AML cells, in vitro studies are still routinely performed in supra-physiologic ambient O2 concentration (21%), with potentially important consequences for clinical translatability. We hereby present an integrated molecular dissection of AML cells response to Cytarabine under O2-controlled conditions and exploit the information to develop novel synergistic/additive treatment combinations.Methods: M14, THP1, OCIAML3 and primary cells (PC) derived from AML patients of different risk groups were incubated in culture medium under 21% O2 or 1% O2 in the presence or absence of Cytarabine (Cy, 1-7.5 µM), Rosuvastatin (Ro, 10-200µM) and/or Pravastatin (Pra, 10-200µM). After 48h, proliferation and apoptosis assays were performed using MTT assays, annexin V/PI staining and FACS analysis. Characterization of gene expression and expression levels of total and phosphorylated proteins were assessed using RNAseq and Reverse Phase Protein Arrays (RPPA), respectively. Gene Set Enrichment Analysis was performed following GSEA User Guide. Quantitative PCR reactions were performed using PowerUp SYBR Green PCR Master Mix (Applied Biosystems, USA) on 7900HT Real-Time PCR System. Intracellular cholesterol (ICC) levels were measured using WAKO Cholesterol assay kit.Results: Treatment with Cy under 1% O2 induced a specific gene signature of metabolic stress responses, including the perturbation of cholesterol homeostasis. Detailed analysis of the cholesterol pathway via RNAseq showed pathway induction by hypoxia itself and severe depletion by Cy. Focused RT-PCR assays on the cholesterol pathway components HMGCS1, MSMO1, LSS and SQLE demonstrated significant induction under 1% compared to 21% O2, and significant downregulation in response to Cy. In line with our RNAseq and PCR data, RPPA showed that critical cholesterol synthesis pathways, such as ACC1, are downregulated in response to Cy. Surprisingly, we observed that ICC levels significantly increased after 48h in response to treatment with Cy (p<.01, n=4 [M14] and [THP1], 21% O2; p<.05, n=4 [M14] and [THP1], 1% O2). In addition, Cy therapy led to strong induction of CD36 mRNA expression (e.g. up to 11.7-fold at 21% O2, p<.05, up to 7.3-fold at 1% O2, p<.01, n=3 [M14]). In order to investigate the effects of cholesterol inhibition against AML cells, we treated several AML cell lines and PCs with Ro or Pra under 21% and 1% O2. We found that Ro exerts dose dependent antileukemic activity at 21% and 1% O2 and, at all doses tested, was significantly more effective than Pra, which was not effective against AML cells at 1% O2. The cytotoxic effects of Ro consisted of lowering ICC levels, induction of apoptosis and inhibition of AML cell growth. Further, Ro combined with Cy significantly enhanced the antileukemic effects of Cy alone and exhibited additive to synergistic activity in AML cell lines (CImax= 0.55, p<.05, n=3) and strong synergy in PCs (CImax= 0.22, n=10) derived from AML patients of different risk groups under 21% and 1% O2. Remarkably, Ro was not toxic against human CD34+ cord blood cells.Summary:Treatment with Cytarabine induces a genetic signature of metabolic stress response, including the perturbation of the cholesterol biosynthesis pathway, in AML cells.Depletion of intracellular cholesterol levels is counterbalanced by upregulation of CD36 expression in Cytarabine-treated AML cells under 21% and 1% O2 conditions.Rosuvastatin exerts antileukemic activity via downregulation of intracellular cholesterol levels, induction of apoptosis and inhibition of AML cell growth.Rosuvastatin displays greater antileukemic effects than pravastatin.Rosuvastatin combined with Cytarabine acts synergistically against AML cells.Conclusions: Further investigation of Rosuvastatin as a component of anti-AML therapy is warranted. DisclosuresNo relevant conflicts of interest to declare.

Novel Therapeutic Targeting of UHRF1 Restores 5'-Azacytidine Response in Resistant Acute Myeloid Leukemia

DNA hypermethylation plays a pivotal role in the pathogenesis of acute myeloid leukemia (AML). Most of the recurrent driver mutations and chromosomal translocations in AML involve genes encoding chromatin modifiers and DNA methylation relevant enzymes. Hypo-methylating drugs such as 5-Azacytidine (AZA) that target DNMTs prolong overall survival in AML patients. However, their long term treatments lead to emergence of acquired therapy resistance mostly through unknown mechanisms and hence there is an urgent need for alternate therapeutics to address AZA resistance in AML patients. Recently, it has been shown that AZA resistant leukemic cells are relatively quiescent with higher expression of many components of DNA methylation machinery that also includes UHRF1 (ubiquitin-like with PHD and ring finger domains 1). UHRF1 is a key epigenetic modulator that regulates DNA methylation and gene expression. It is a multi-domain nuclear protein with an SRA (SET-and-RING-associated) domain to recognize hemi-methylated DNA immediately after replication. It plays a crucial role in the maintenance of DNA methylation by recruiting DNMT1 to replication sites and facilitates methylation on newly synthesized DNA strand. UHRF1 is frequently overexpressed in multiple human neoplasms including AML and in the absence of UHRF1, hematopoietic stem cells undergo erythroid-biased differentiation at the expense of self-renewal capacity. Despite UHRF1 being key a therapeutic target against AML, specific, and cell-permeable inhibitors of UHRF1 have not been identified yet.In this study, we hypothesized that targeting UHRF1 using novel small molecule inhibitor will interfere with DNMT1-dependent DNA methylation at newly synthesized DNA strand, which may further synergize with antiproliferative effect of classical DNMT inhibitors in AML cells. In this study, we used in silico strategy to discover novel putative UHRF1 inhibitors by screening NCI compound database. For in vitro validation, we have first purified the SRA domain of UHRF1 followed by analysis of total DNA methylation levels using 5'-methyl cytosine (5mC) dot blot in the presence of each inhibitor. After a series of stringent in vitro and cell based assays we have identified lead compound 20 (C20) as a potent UHRF1 inhibitor which suppresses DNA methylation without affecting DNMTs in leukemic cells. Specificity of C20 against SRA domain was further established by isothermal titration calorimetry (ITC). We next found that C20 treatment significantly decreased UHRF1 and DNMT1 foci formation in the nucleus of mouse embryonic fibroblast and stem cells. Based on the its critical role in DNA methylation and enhanced expression in resistant cells, we assumed that AZA resistance in AML may be mediated by UHRF1 and C20 might restore AZA sensitivity by attenuating enhanced UHRF1 activity. To validate this, we pretreated AZA resistant leukemic cells (HL60R) with suboptimal dose of C20 followed by AZA treatment. Interestingly, we found a synergistic increase in antiproliferative effect by flow cytometry and colony formation assay. By analyzing the surface expression of myeloid differentiation markers, we found that C20 treatment promotes differentiation and decreases quiescent leukemic cell population. In conclusion, we report a novel UHRF1 inhibitor as a sensitizer of resistant AML cells towards AZA treatment potentially by promoting differentiation, suggesting a novel combination approach for future clinical evaluations. DisclosuresNo relevant conflicts of interest to declare.

Combined Targeting of BET Family Proteins and BCL2 Is Synergistic in Acute Myeloid Leukemia Cells Overexpressing S100A8 and S100A9

BackgroundThe 5-year survival rate for acute myeloid leukemia (AML) remains poor with most patients succumbing to relapse or refractory disease. Recently, the BCL2 specific inhibitor venetoclax has shown promising anti-leukemia activity in high-risk AML patients. Most patients, however, ultimately develop resistance to monotherapy and novel combination treatments with venetoclax are needed for patients with no other therapy options available. In this study we identified high expression of calcium binding protein S100A8/S100A9 to be associated with venetoclax resistance and looked for drug combinations to overcome the resistance in AML patient samples overexpressing S100A8 and S100A9.MethodsGene expression was assessed by RNA sequencing of AML patient samples and validated by qPCR. Gene enrichment analysis was performed on differentially expressed genes between venetoclax highly sensitive (n=3) and resistant (n=4) samples. Sensitivity of AML patient derived mononuclear cells was assessed to 304 different small molecule inhibitors by measuring cell viability after 72 h incubation with 5 different concentrations (1-10,000 nM) using the CellTiter-Glo (CTG) assay. A drug sensitivity score (DSS) was calculated based on a modified area under the dose response curve calculation. Drug combination studies were performed using AML cell lines resistant to venetoclax and confirmed with primary patient cells (n=15). Data of the drug combination studies were analyzed with the Zero Interaction Potency (ZIP) model by considering a dose-response matrix where two drugs are tested at 8 concentrations in a serially diluted manner. Statistical dependence between gene expression and drug sensitivity data was assessed by Pearson's correlation coefficient modelling.ResultsVenetoclax resistant AML patient samples were found to overexpress genes related to immune responses including inflammatory related proteins S100A8 and S100A9. The expression of S100A8 and S100A9 was upregulated in a sub-group of AML patients with somatic mutations in DNA methylation genes IDH2 and TET2 and chromatin modifier ASXL1. Functional studies with AML cell lines validated high expression of the S100 proteins in cells insensitive to venetoclax (NOMO-1, SKM-1 and SHI-1) whereas sensitive cell lines (MOLM-13, Kasumi-1 and ML-2) did not express the proteins. Integrated analysis of S100A8 and S100A9 expression and ex vivo drug sensitivity data indicated positive correlation of S100 expression with sensitivity to BET inhibitor (birabresib), PI3K inhibitor (TG100-115) and MEK1/2 inhibitor (AZD8330). In contrast, sensitivity to venetoclax and the FLT3 inhibitor quizartinib negatively correlated with S100 gene expression. Subsequently, we combined positively correlating drugs with venetoclax and tested the efficacy of these combinations in AML cell lines and patient samples. From the drug combination studies we found that BET inhibitor birabresib was able to overcome resistance to venetoclax treatment. The BCL2/BET inhibitor combination was synergistic in venetoclax resistant cell lines NOMO-1 and SKM-1, which express high-levels of S100A8 and S100A9 (Figure 1A). Efficacy of the combination on primary AML patient samples correlated with the expression level of the S100 genes. Nine of 11 high expression samples were sensitive to the venetoclax/birabresib combination (Figure 1B-C), whereas no synergy was observed in 3 of 4 samples with a low level of S100 expression.ConclusionsThe calcium binding proteins S100A8 and S100A9 are abundant in myeloid cells and are associated with poor prognosis in AML (Edgeworth et al, J Biol Chem. 1991; Nicolas et al, Leukemia 2011). From ex vivo and in vitro analyses of AML, we found that high expression of S100A8 and S100A9 is highly correlative with resistance to the BCL2 inhibitor venetoclax. In contrast, high S100A8 and S100A9 expression correlates with sensitivity to BET inhibitor birabresib. Interestingly, our studies showed that these two drugs act synergistically in venetoclax resistant AML cell lines and AML patient samples and may be a beneficial novel combination that should be further confirmed in preclinical and clinical investigations. [Display omitted] DisclosuresPorkka:Celgene: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Heckman:Orion Pharma: Research Funding; Novartis: Research Funding; Celgene: Research Funding.

Paradox-Breaker Pan-RAF Inhibitors Induce an AML-Specific Cytotoxic Response and Synergize with Venetoclax to Display Superior Antileukemic Activity

The RAS-RAF-MEK-ERK (MAPK) pathway is deregulated in many cancers including acute myeloid leukemia (AML), where it facilitates cancer progression, chemo-resistance and cancer relapse. Therefore, the inhibition of this pathway has been explored for the treatment of AML, especially in the ~10% of AML cases where the leukemic cells carry activating mutations of the NRAS or KRAS oncogenes. However, MEK inhibitors, the best explored class of MAPK inhibitors, have not proven effective in the clinical setting. A novel clinically investigated drug class in this context are the paradox-breaker pan-RAF inhibitors that prevent the reactivation of the MAPK-pathway and have exhibited single agent efficacy in preclinical testing for solid cancers. Therefore, it is relevant to explore the utility of this class of drugs for AML treatment.We found that the paradox breaker pan-RAF inhibitors LY3009120 (currently in clinical testing), AZ-628 and TAK-632 as well as other MAPK inhibitors effectively downregulated MAPK signaling activity (ERK phosphorylation) in AML cells. However, when comparing cell viability (CellTiter Glo) and cell death (CellTox Green) responses in 101 AML patient sample cohort, only the pan-RAF inhibitors induced cytotoxicity while the other MAPK-pathway inhibitors generally induced a temporary cytostatic response. Specifically, LY3009120 induced cell death in 42 samples out of 101 (42 %) whereas only 9/101 (9 %) underwent cell death in response to trametinib (MEK-inhibitor) (Fig. panel A). Importantly, LY3009120 did not induce cell death in mononuclear bone marrow cells obtained from healthy individuals (5 samples) or in non-hematopoietic cell lines (Fig. panel A).To get insight into the mechanism regulating cell death in response to pan-RAF inhibition we first characterized that cells underwent rapid caspase-3/7 mediated apoptotic cell death in response to pan-RAF inhibitors (Fig. panel B). Therefore, we analyzed the proteins involved in apoptosis pathways and found that MCL-1 protein, but not mRNA levels, were downregulated upon treatment with pan-RAF inhibitors but not with MEK-inhibitors. This suggests that pan-RAF inhibition negatively regulated the translation of MCL-1, thus uncovering a new mode of MCL-1 regulation by RAF kinases. Importantly, pan-RAF inhibitors downregulated MCL-1 levels independently of caspase activity, indicating that MCL-1 downregulation precedes the apoptotic response.We explored the link between somatic mutation status and response to LY3009120 in our AML patient sample cohort. KRAS, KIT, FLT3-TKD, BCOR, JAK2 and PTPN11 mutations were found to correlate positively with pan-RAF inhibitor response, while SRSF2, RUNX1, GATA2 and STAG2 mutations negatively correlated with response and appeared to override positively correlating mutations.As pan-RAF inhibition downregulates MCL-1, an important survival protein whose increased expression is known to cause resistance to BCL-2 inhibitors, we hypothesized that inhibition of RAF kinases can overcome the resistance to BCL-2 inhibitors. In order to test the hypothesis, we generated venetoclax (BCL-2 inhibitor) resistant MOLM-13 cells and treated them with single and combined doses of venetoclax and LY3009120. As expected, combined inhibition of BCL-2 and RAF kinases significantly increased cytotoxicity as compared to either of the drugs alone (Fig. panel C). Conversely, to see if BCL-2 inhibition can overcome resistance to pan-RAF inhibitors, we treated pan-RAF inhibitor resistant HL-60 and THP-1 cells with single and combined doses of venetoclax and LY3009120. Interestingly, the drug combination exhibited synergistic cytotoxicity in HL-60 cells (where LY3009120 alone downregulates MCL-1) but not in THP-1 cells (where LY3009120 alone does not downregulate MCL-1). This indicates that MCL-1 downregulation upon pan-RAF inhibitor treatment is critical for the observed combination cytotoxicity.In summary, our results show that paradox breaker pan-RAF inhibitors are effective as single agents to induce cell death in AML blasts, likely through a combined MAPK pathway and MCL-1 dependent mechanism. Moreover, combined inhibition of RAF kinases and BCL-2 can overcome resistance of AML cells to either drug alone and lead to superior anti-leukemic activity. [Display omitted] DisclosuresHeckman:Celgene: Research Funding; Novartis: Research Funding; Orion Pharma: Research Funding. Porkka:Novartis: Honoraria, Research Funding; Celgene: Honoraria, Research Funding. Wennerberg:Novartis: Research Funding.

CircPAN3 mediates drug resistance in acute myeloid leukemia through the miR-153-5p/miR-183-5p–XIAP axis

The contribution and role of circular RNAs (circRNAs) in mediating chemoresistance in acute myeloid leukemia (AML) are still poorly understood and need further investigation. In this study, we established a doxorubicin (ADM)-resistant THP-1 AML cell line (THP-1/ADM). A high-throughput microarray was used to identify circRNA expression profiles of THP-1/ADM cells and naive THP-1 cells. The identified potential functional circRNA molecule was further validated in THP-1/ADM cells and bone marrow (BM) specimens from 42 AML patients. The interactions with target microRNAs (miRNAs) and downstream messenger RNAs (mRNAs) were also explored. As a result, 49 circRNAs that are significantly differentially expressed between THP-1/ADM and THP-1 cells were identified. Of these circRNAs, downregulation of circPAN3 by small interfering RNA significantly restored ADM sensitivity of THP-1/ADM cells. Furthermore, BM samples from patients with refractory and recurrent AML showed increased expression of circPAN3. A detailed circRNA/miRNA/mRNA interaction network was predicated for this circRNA. Subsequent mechanistic experiments showed that downregulation of circPAN3 could decrease the expression of X-linked inhibitor of apoptosis protein (XIAP), but this effect was counteracted by miR-153-3p or miR-183-5p specific inhibitors. Luciferase experiments further demonstrated that these molecules are involved in the circPAN3 regulatory network. Our results revealed that circPAN3 may be a key mediator for chemoresistance of AML cells, which may depend on the circPAN3-miR-153-5p/miR-183-5p-XIAP axis. Our findings provide evidence that circPAN3 can be a valuable indicator for predicting clinical efficacy of chemotherapy in AML patients and also can serve as a potential target for reversing drug resistance in AML.

TLR4/NF-κB axis induces fludarabine resistance by suppressing TXNIP expression in acute myeloid leukemia cells

Overcoming drug resistance is one of key issues in treating refractory acute myeloid leukemia (AML). The Toll-like receptor 4 (TLR4) signaling pathway is involved in many aspects of biological functions of AML cells, including the regulation of pro-inflammatory cytokine products, myeloid differentiation, and survival of AML cells. Thus, targeting TLR4 of AML patients for therapeutic purposes should be carefully addressed. In this regard, we investigated the possible role of TLR4 as a regulatory factor against fludarabine (FA) cytotoxicity activity. Here, we identified the differential expression of TLR4 and CD14 receptors in AML cell lines and examined their relationship to FA sensitivity. We found that the stimulation of TLR4 with lipopolysaccharide (LPS) in a TLR4-expressing cell line, THP-1, increased cell viability under FA treatment condition and showed that TLR4 stimulation overcame FA sensitivity through the activation of NF-κB, which subsequently upregulated several anti-apoptotic genes. The inhibition of TLR4/NF-κB signaling could partially or completely reverse LPS-induced cell survival under FA treatment conditions. Interestingly, we found that the expression of thioredoxin-interacting protein (TXNIP), a well-known tumor suppressor, was induced by FA treatment; however, it was suppressed by LPS treatment. Furthermore, the expression level of TXNIP was critical for FA-induced cytotoxicity or LPS-induced FA resistance of THP-1 cells. Our data suggest that TXNIP plays an important role in FA-induced cytotoxicity and TLR4/NF-κB-mediated FA resistance of AML cells. Therefore, TXNIP may be a potential therapeutic target for AML treatment.

P-131 - γ-H2AX, CYP3A and CYP2J2 overexpression in a drug-resistant acute myeloid leukemia cell line

Drug resistance is a major clinical problem in cancer treatment. Thus, it is important to establish cell line models to better understand and counteract this problem. The aim of this study was to establish and characterize a drug resistant acute myeloid leukemia cell line, as a sub-clone from a drug sensitive parental cell line. The resistant cell line (HL60–734 VR) was established by treating the parental sensitive cell line (HL60) with increasing concentrations of doxorubicin during several months. Dose-response curves were performed to confirm that the HL60–734 VR cell line was resistant to doxorubicin and to three other drugs (etoposide, cisplatin and daunorubicin). The resistant phenotype was not lost upon drug removal for 1 month. Resistant cells did not express the drug efflux pumps P-gp or BCRP, neither had alterations in apoptotic proteins such as Bax, XIAP, p53, Caspase-3 and Bcl-2. Resistant cells showed alterations in cell cycle profile and presented increased expression of a DNA repair protein (γ-H2AX) and also of proteins involved in drug metabolism (CYP3A and CYP2J2), suggesting that these alterations might be associated with the drug resistant phenotype in these cells.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Emodin and Its Combination with Cytarabine Induce Apoptosis in Resistant Acute Myeloid Leukemia Cells in Vitro and in Vivo

Background/Aims: Acute myeloid leukemia (AML) remains a hematologic malignancy with poor survival and a high risk of relapse, which is mainly caused by the emergence of multidrug resistance (MDR). The identification of novel agents to improve therapeutic strategies becomes important priority for AML treatment. It has been shown that emodin has therapeutic effects on many kinds of human malignant tumors. In this study, we investigated the anti-leukemia effects of emodin alone or in combination with cytarabine (Ara-C) on multidrug-resistant AML HL-60/ADR cells and in a mouse xenograft model of human highly tumorigenic AML HL-60/H3 cells. The underlying mechanism was also addressed. Methods: Cell viability after treatment was measured by MTT assay. The DNA fragmentation assay, Annexin V-PE/7-AAD, AO/EB staining, and electron microscopy were introduced to assess the apoptotic induction effects. Changes in protein expression in the Akt and ERK signaling pathways were determined by western blotting. In vivo antileukemia effects on HL-60/H3 xenograft model and overall mouse survival outcomes were further analyzed in this study. Results: Emodin dose-dependently induced growth inhibition and apoptotic effects in resistant HL-60/ADR cells in vitro as well as in the HL-60/H3 xenograft models in vivo. Moreover, emodin significantly enhanced chemosensitivity of AML cells to Ara-C, inhibited leukemic cell growth, and improved survival in the mouse xenograft model of AML. Dual targeting of Akt and ERK signaling pathways might contribute to the anti-leukemia effects on AML cells in vitro and in vivo. Conclusion: Emodin and its combination with Ara-C may be considered a promising therapeutic approach in AML and worthy of further investigation.

Knockdown of Long Noncoding RNA HOXA-AS2 Suppresses Chemoresistance of Acute Myeloid Leukemia via the miR-520c-3p/S100A4 Axis

Background/Aims: Among different molecular candidates, there is growing data to support that long noncoding RNAs (lncRNAs) play a significant role in acute myeloid leukemia (AML). HOXA-AS2 is significantly overexpressed in a variety of tumors and associated with anti-cancer drug resistance, however, little is known regarding the expression and function of HOXA-AS2 in the chemoresistance of AML. In this study, we aimed to determine the role and molecular mechanism of HOXA-AS2 in adriamycin-based chemotherapy resistance in AML cells. Methods: Quantitative real-time PCR was used to detect HOXA-AS2 expression in the BM samples and ADR cell lines, U/A and T/A cells. Furthermore, the effects of HOXA-AS2 silencing on cell proliferation and apoptosis were assessed in vitro by CCK8 and flow cytometry, and on tumor growth in vivo. Furthermore, bioinformatics online programs predicted and luciferase reporter assay were used to validate the association of HOXA-AS2 and miR-520c-3p in AML. Results: In this study, we showed that HOXA-AS2 is significantly upregulated in BM samples from AML patients after treatment with adriamycin-based chemotherapy and in U/A and T/A cells. Knockdown of HOXA-AS2 inhibited ADR cell proliferation in vitro and in vivo and promoted apoptosis. Bioinformatics online programs predicted that HOXA-AS2 sponge miR-520c-3p at 3’-UTR with complementary binding sites, which was validated using luciferase reporter assay and anti-Ago2 RIP assay. HOXA-AS2 could negatively regulate the expression of miR-520c-3p in ADR cells. S100A4 was predicted as a downstream target of miR-520c-3p, which was confirmed by luciferase reporter assay. Conclusion: Our results suggest that HOXA-AS2 plays an important role in the resistance of AML cells to adriamycin. Thus, HOXA-AS2 may represent a therapeutic target for overcoming resistance to adriamycin-based chemotherapy in AML.