Resistant Leukemia Research Articles

H2O2 mediated FLIP and XIAP down-regulation involves increased ITCH expression and ERK-Akt crosstalk in imatinib resistant Chronic Myeloid Leukemia cell line K562

Regulation of anti-apoptotic protein FLICE-like inhibitory protein (FLIP) and X-linked inhibitor of apoptosis protein (XIAP) remains a crucial step in the cell fate determination and thus targeting these anti-apoptotic proteins could be a viable strategy for the treatment of cancer. However the regulation of FLIP and XIAP is not very well established till date. Here we have shown that ROS decreased XIAP and FLIP by activation of ubiquitin-proteasomal pathway in imatinib resistant K562 cells. Activation of the components of MAPK pathway, ERK and JNK, played a crucial role in XIAP and FLIP degradation because ectopic expression or knock down of ERK and JNK changed the pattern of ROS mediated down-regulation of these two proteins. We have also found that JNK and ERK differentially regulates FLIP and XIAP, respectively. Moreover, our data suggests that activated ERK decreased Akt phosphorylation and thus its binding to and stabilization of XIAP. On the other hand, JNK activation increased E3 ubiquitin ligase ITCH expression and its binding to FLIP which leads to its degradation. Thus, we have, for the first time elucidated that ROS mediated ERK-Akt crosstalk regulates XIAP. We have also shown for the first time that ROS regulates ITCH expression which controls FLIP degradation.

In Vitro Modulation of P-Glycoprotein Activity by Euphorbia intisy Essential Oil on Acute Myeloid Leukemia Cell Line HL-60R.

Euphorbia species have a large spectrum of traditional medicinal uses. We tested the biological activities of the essential oil (EO) of Euphorbia intisy Drake in an acquired multidrug resistance leukemia model to assess whether the EO obtained by hydrodistillation of stems was able to reverse the resistant phenotype. HL-60R cell lines are characterized by the overexpression of P-glycoprotein (P-gp), inhibitors of apoptosis proteins (IAPs) and constitutive expression of NF-κB. EO chemical composition was determined by GC/MS analysis; cytotoxic activity of EO by MTS assay alone or in combination with doxorubicin; pro-apoptotic effect and doxorubicin accumulation were analyzed by flow cytometry; P-gp ATPase activity was measured by P-gp-Glo™ assay systems kit. The ability to inhibit NF-κB and its target genes was also assessed. E. intisy EO exhibited a comparable cytotoxic effect and ability to block P-gp in both the HL-60 and its MDR variant HL-60R. In addition, EO suppressed P-gp protein expression and significantly downregulated MDR1 mRNA level, as well as some IAPs proteins, probably through the inhibition of NF-κB. Our results suggest that E. intisy EO could reverse P-gp-mediated drug resistance in tumor cells acting as a chemosensitizing agent.

The AML Microenvironment Catalyzes a Step-Wise Evolution to Gilteritinib Resistance

Our study details the stepwise evolution of gilteritinib resistance in FLT3-mutated acute myeloid leukemia (AML). Early resistance is mediated by the bone marrow microenvironment, which protects residual leukemia cells. Removing these supportive extrinsic ligands drives evolution of late, intrinsic resistance. Whole exome sequencing, CRISPR/Cas, metabolomics, proteomics, and pharmacologic approaches were used to mechanistically define both early and late resistance. Early resistant cells undergo metabolic reprogramming, grow more slowly, and are dependent upon Aurora kinase B (AURKB). Late resistant cells are characterized byexpansion of pre-existing NRAS mutant subclones and continued metabolic reprogramming. Our model closely mirrors the timing and mutations of AML patients treated with gilteritinib.Pharmacological inhibition of AURKB resensitized both early resistant cell cultures and primary leukemia cells from gilteritinib-treated AML patients. These findings support a combinatorial strategy to target early resistant AML cells with AURKB inhibitors and gilteritinib before the expansion of pre-existing resistance mutations occurs.

Investigation of a new oxazolidine derivative in human resistance acute leukemia cells: deciphering its mechanism of action by label-free proteomic.

The present study aimed to evaluate the mechanism of action of the antineoplastic activity of an oxazolidine derivative, LPSF/NB-3 (5-(4-cloro-benzilideno)-3-etil-2-tioxo-oxazolidin-4-ona). Cytotoxicity assays were performed in peripheral blood mononuclear cells (PBMCs) and resistant acute leukemia cell line (HL-60/MX1) by the MTT method. LPSF/NB-3 exhibited cytotoxicity in HL-60/MX1, but it was not toxic to healthy cells in the highest dose tested (100 μM). The protein extract of HL-60/MX1 cells treated with LPSF/NB-3 was subjected to proteomic analysis using two-dimensional chromatography coupled to mass spectrometry. We could identify a total of 2652 proteins, in which 633 were statistically modulated. Within the group of protein considered for the quantitative analysis with the established criteria, 262 were differentially expressed, 146 with increased expression and 116 with decreased expression in the sample treated with LPSF/NB-3 compared to the control. The following differentially expressed pathways were found: involving regulation of the cytoskeleton, DNA damage, and transduce cellular signals. Networks that were highlighted are related to the immune system. The ELISA technique was used to assess the immunomodulatory potential of LPSF/NB-3 in PBMCs. We observed significant decrease of IFNγ (p < 0.01) and dose-response pattern of the cytokines IL-6, IL-17A, IL-22, and IL-10. Therefore, results suggest that LPSF/NB-3 appears to modulate important pathways, including cell cycle and immune system regulatory pathways.

Targeting USP47 overcomes tyrosine kinase inhibitor resistance and eradicates leukemia stem/progenitor cells in chronic myelogenous leukemia

Identifying novel drug targets to overcome resistance to tyrosine kinase inhibitors (TKIs) and eradicating leukemia stem/progenitor cells are required for the treatment of chronic myelogenous leukemia (CML). Here, we show that ubiquitin-specific peptidase 47 (USP47) is a potential target to overcome TKI resistance. Functional analysis shows that USP47 knockdown represses proliferation of CML cells sensitive or resistant to imatinib in vitro and in vivo. The knockout of Usp47 significantly inhibits BCR-ABL and BCR-ABLT315I-induced CML in mice with the reduction of Lin−Sca1+c-Kit+ CML stem/progenitor cells. Mechanistic studies show that stabilizing Y-box binding protein 1 contributes to USP47-mediated DNA damage repair in CML cells. Inhibiting USP47 by P22077 exerts cytotoxicity to CML cells with or without TKI resistance in vitro and in vivo. Moreover, P22077 eliminates leukemia stem/progenitor cells in CML mice. Together, targeting USP47 is a promising strategy to overcome TKI resistance and eradicate leukemia stem/progenitor cells in CML.

Myeloid/lymphoid neoplasms with eosinophilia: clinical picture and therapeutic approaches

Myeloid/lymphoid neoplasms with eosinophilia (M/Leo) and tyrosine kinase (TK) fusion genes constitute a separate category within the 2016 World Health Organization (WHO) classification. All these are characterized by blood or tissue eosinophilia and the presence of a unique genetic abnormality. M/Leo may have diverse clinical manifestations with variable response to TK inhibitors (TKI). PDGFRA -rearranged neoplasms (usually with detectable FIP1L1-PDGFRA ) are found to be extremely sensitive to low dose of imatinib (IM at 100 mg daily) with nearly 100% hematological complete response rate. Moreover, >90% of IM treated patients may achieve long-term molecular response. IM discontinuation may result in sustained remission in c.50–60% of patients. An excellent response to IM (but at 400 mg/day) was also demonstrated for patients with PDGFRB rearrangements, but trials on IM cessation were not attempted. The FGFR1 -rearranged neoplasms are associated with an aggressive disease course and allogeneic stem cell transplantation (allo-SCT) is the only potentially curative approach. Participation in clinical trials should be recommended. Recently, pemigatinib was found to be effective in a proportion of FGFR1 -rearranged individuals. An aggressive outcome with rapid blast transformation is also characteristic for the JAK2 -rearranged neoplasms. These patients should be included in clinical trials or attempted with ruxolitinib or fedratinib as a ‘bridge’ to allo-SCT. A new category of neoplasms with eosinophilia and FLT3 and ABL1 rearrangements has not yet been incorporated into the WHO 2016 classification. The prognosis is poor with a tendency to evolve into resistant acute leukemia. The treatment includes TKI with known activity against FLT3/ABL1 followed by allo-SCT.

Synthesis, characterization and biological activity of bis[3-ethyl-4-aryl-5-(2-methoxypyridin-5-yl)-1-propyl-1,3-dihydro-2H-imidazol-2-ylidene]gold(i) complexes.

A series of bis[3-ethyl-4-aryl-5-(2-methoxypyridin-5-yl)-1-propyl-1,3-dihydro-2H-imidazol-2-ylidene]gold(i) complexes (2a-f) containing methyl, fluoro or methoxy substituents at various positions in the 4-aryl ring was synthesized and evaluated for their anti-cancer properties in A2780 (wild-type and Cisplatin-resistant) ovarian carcinoma as well as LAMA 84 (imatinib-sensitive and -resistant) and HL-60 leukemia cell lines. The bis-NHC gold(i) complexes were more active compared to their related mono-NHC gold(i) analogues and reduced proliferation and metabolic activity in a low micromolar range. With the exception of 2d (3-F), the compounds displayed higher potency than the established drugs Auranofin and Cisplatin. The lack of effects against non-cancerous lung fibroblast SV-80 cells indicated a high selectivity towards tumor cells. All tested complexes generated reactive oxygen species in A2780cis cells; however, the induction of apoptosis was very low. Furthermore, thioredoxin reductase is not the main target of these complexes, because its inhibition pattern did not correlate with their biological activity.

Physiological properties and characteristic reactions of hydroxyurea

As it was mentioned in the previous paper, we observed the mechanism of action the interesting drug, first synthesized back in 1869 for the first time, called Hydroxyurea. A century later, phase I and II trials began to test its safety in humans with solid tumors. It was first approved by the FDA in 1967 for the treatment of neoplastic diseases and is presently approved for the treatment of melanoma, resistant chronic myelocytic leukemia (CML), and recurrent, metastatic testicular and ovarian cancer. Sickle cell disease is a genetic disorder that decreases life expectancy by 25 to 30 years. Individuals are diagnosed with sickle cell disease if they have one of several genotypes that result in at least half of their hemoglobin being hemoglobin S (HbS). Sickle cell anemia refers specifically to the condition associated with homozygosity for the Hb S mutation (Hb SS). Several other hemoglobin mutations, when occurring with an Hb S mutation, cause a similar but often milder disease than sickle cell anemia. In addition to reduced life expectancy, patients with sickle cell disease experience chronic pain and reduced quality of life. Painful crises, also known as vaso-occlusive crises, are the most common reason for emergency department use and hospitalization, and acute chest syndrome is the most common cause of death. Prior to the approval of hydroxyurea for use in sickle cell disease, patients with this condition were treated only with supportive therapies. These measures included penicillin in children to prevent pneumococcal disease, routine immunizations, and hydration and narcotic therapy to treat painful events. Red blood cell transfusions increase the blood’s oxygen carrying capacity and decrease the concentration of cells with abnormal hemoglobin, but chronic transfusion therapy predictably leads to iron overload and alloimmunization. Therapies such as hydroxyurea that raise fetal hemoglobin (Hb F, α2γ2) levels are promising because they effectively lower the concentration of Hb S within a cell, resulting in less polymerization of the abnormal hemoglobin.Hydroxyurea’s efficacy in sickle cell disease is generally attributed to its ability to raise the levels of Hb F in the blood; however, the mechanisms by which it does so are unclear. Early studies suggested that hydroxyurea is cytotoxic to the more rapidly dividing late erythroid precursors, resulting in the recruitment of early erythroid precursors with an increased capacity to produce HbF.

Molecular Remission in PML/RARα-Positive Acute Promyelocytic Leukemia by Combined All-trans Retinoic Acid and Idarubicin (AIDA) Therapy

AbstractTwo hundred fifty-three patients with newly diagnosed acute promyelocytic leukemia (APL) were eligible to enter the multicentric GIMEMA-AIEOP “AIDA” trial during the period July 1993 to February 1996. As a mandatory prerequisite for eligibility, all patients had genetic evidence of the specific t(15; 17) lesion in their leukemic cells confirmed by karyotyping or by reverse transcription-polymerase chain reaction (RT-PCR) of the PML/RARα fusion gene (the latter available in 247 cases). Median age was 37.8 years (range, 2.2 to 73.9). Induction treatment consisted of oral all-trans retinoic acid (ATRA), 45 mg/m2/d until complete remission (CR), given with intravenous Idarubicin, 12 mg/m2/d on days 2, 4, 6, and 8. Three polychemotherapy cycles were given as consolidation. Hematologic and molecular response by RT-PCR was assessed after induction and after consolidation. At the time of analysis, 240 of the 253 eligible patients were evaluable for induction. Of these, 11 (5%) died of early complications and 229 (95%) achieved hematologic remission. No cases of resistant leukemia were observed. Of 139 cases studied by RT-PCR after induction, 84 (60.5%) were PCR-negative and 55 (39.5%) PCR-positive. One hundred sixty-two patients were evaluable by RT-PCR at the end of consolidation. Of these, 159 (98%) tested PCR-negative and 3 (2%), PCR-positive. After a median follow up of 12 months (range, 0 to 33), the estimated actuarial event-free survival for the whole series of 253 eligible patients was 83% ± 2.6% and 79% ± 3.2% at 1 and 2 years, respectively. This study indicates that the AIDA protocol is a well-tolerated regimen that induces molecular remission in almost all patients with PML/RARα-positive APL. Preliminary survival data suggest that a remarkable cure rate can be obtained with this treatment.

Point: Is there a best duration of deep molecular response to achieve therapy-free remission in chronic myeloid leukaemia?

Point: Is there a best duration of deep molecular response to achieve therapy-free remission in chronic myeloid leukaemia?

Co-Targeting BET Bromodomain Proteins and Aberrant Signaling in AML

AML remains a therapeutic challenge with a high mortality rate, underscoring the need for new biologically based therapies. Somatic alterations that deregulate epigenetic programs and signal transduction pathways frequently coexist in AML. While the former class of alterations is hypothesized to promote a chromatin state that is permissive for AML development and essential for leukemia maintenance, experimental data also suggest that signaling mutations play a central role in driving leukemic growth. Thus, simultaneously targeting the abnormal epigenetic landscape and aberrant signaling is a rational therapeutic approach. We previously used the MEK inhibitor PD0325901 (PD901) to target the Raf/MEK/ERK (MAPK) effector pathway in primary murine AMLs in vivo. Here we report substantial preclinical efficacy of the BET inhibitor PLX51107 in several independent primary AMLs that was further enhanced by PD901. We first exposed five NRAS or KRAS mutant human AML cell lines to varying concentrations of PLX51107 and PD901. These studies revealed potent synergy based on cell proliferation and apoptosis readouts (Figure 1A). We next performed RNA-seq on the NRAS-mutant OCI-AML3 cell line treated with PD901, PLX51107, or the combination. Remarkably, PLX51107 + PD901 cooperated to potently downregulate MYC and its transcriptional targets (Figure 1B). Additionally, AML cells increased the expression of p53 transcriptional targets in response to PLX51107 and CRISPRi-mediated knock-down of TP53 expression abrogated the synergistic effects of PLX51107 + PD901 (Figure 1C). We previously performed retroviral insertional mutagenesis in NrasG12D mice to generate genetically diverse panels of transplantable, primary AMLs. To follow-up on our promising in vitro data, we transplanted 5 of these leukemias into cohorts of recipient mice and treated them with PLX51107 ± PD901. PLX51107 (10 mg/kg/day) showed impressive efficacy that was further enhanced by a modest dose of PD901 (1.5 mg/kg given 4 days per week; Figure 1D). To begin characterizing mechanisms of resistance, we evaluated matched pairs of drug-sensitive ("parental") and relapsed leukemias that emerged during drug treatment. We selected relapsed leukemias that exhibited clonal evolution based on distinct novel retroviral integrations for in depth evaluation. Re-transplanting these AMLs into secondary recipients and re-treating them with PLX51107 ± PD901 confirmed intrinsic drug resistance, which was further validated ex vivo in dose response methylcellulose assays (Figure 1D). Remarkably, several of these resistant leukemias down-regulated basal Myc protein expression, which supports the idea that their transcriptional networks are "rewired" (Figure 1E). We next compared basal and dynamic transcriptional profiles in parental and resistant leukemias by exposing them to PLX51107 ex vivo and then performing RNA-seq analysis. Intriguingly, PLX51107 treatment of parental AMLs resulted in transcriptional changes consistent with myeloid maturation in a dose dependent manner (Figure 1E), suggesting that BET inhibition induces differentiation in these leukemias. By contrast, paired resistant AMLs are relatively "immature" compared to their parental counterparts (Figure 1E), which supports this idea that in vivo treatment with PLX51107 selects for the outgrowth of clones at an earlier progenitor state. These resistant AMLs also upregulate Myc transcriptional targets and unexpectedly downregulate Ras transcriptional targets (Figure 1E). We corroborated these findings by analyzing large pediatric AML transcriptome datasets generated as part of the NCI TARGET initiative (Figure 1F). Taken together, our data support the hypothesis that BET inhibitor and MEK inhibitor combination may be a particularly effective therapeutic approach in AML. Additional preclinical testing in patient derived xenograft models is underway. Figure 1 Disclosures Burgess: Bristol Myers Squibb: Current Employment. Severson:Plexxikon Inc.: Current Employment. Powell:Plexxikon Inc.: Current Employment. Bollag:Plexxikon Inc.: Current Employment.

Imetelstat Inhibits Telomerase and Prevents Propagation of ADAR1-Activated Myeloproliferative Neoplasm and Leukemia Stem Cells

BACKGROUND Clonal stem cell derived myeloproliferative neoplasms (MPNs) have a propensity to evolve to acute myeloid leukemia (AML). Deregulation of the innate immune deaminase associated with RNA1 (ADAR1) has been linked to malignant progression and therapeutic resistance. Increased expression of the stem cell gene, human telomerase reverse transcriptase (hTERT), has also been linked with malignant transformation. However, the combinatorial role of ADAR1 and hTERT in the evolution of MPN stem cells to therapy resistant acute myeloid leukemia stem cells (LSCs) and the capacity of a telomerase inhibitor, imetelstat, to prevent survival and self-renewal of pre-LSC and LSC had not been established. Recent clinical trials show early signs of efficacy of imetelstat in treatment of myelofibrosis (MF). However, its role in selectively inhibiting pre-LSC transformation to self-renewing LSC has not been elucidated. Here we show that targeting telomerase activity prevents pre-LSC and LSC maintenance both in vitro and in vivo, suggesting telomerase inhibition as an effective strategy for preventing MPN progression. METHODS To quantify hTERT level and ADAR1 activity in the setting of normal HSPC and MPN stem cell evolution, whole genome sequencing (WGS) analysis was performed on 76 normal and MPN blood CD34+ cells and matching saliva samples. Results were compared with RNA-seq of 100 FACS purified young, aged, MPN and AML CD34+CD38- stem cells and CD34+CD38+ progenitor cells. Confocal fluorescence microscopic evaluation of stem cell ADAR1 and hTERT localization, telomere length by Flow-FISH and telomerase activity by TRAP assays, lentiviral ADAR1 overexpression and shRNA knockdown were performed. In vitro stromal co-cultures, and humanized immunocompromised mouse models were established to determine the impact of imetelstat (a oligonucleotide inhibitor of telomerase) on normal, MPN stem cell and LSC maintenance. RESULTS Combined hTERT overexpression, ADAR1 activation and a significant reduction in telomere length correlated with accelerated stem cell aging during MPN progression to AML. Increased ADAR1 mediated adenosine to inosine (A-to-I) transcript editing coincided with accelerated telomere shortening in high risk MPN stem cells. Moreover, lentiviral ADAR1 overexpression enhanced pre-LSC engraftment. Treatment with imetelstat reduced MPN stem cell and LSC propagation in stromal co-cultures as well as in humanized mouse models commensurate with reduced hTERT expression levels and telomerase activity and decreased ADAR1 editing activity. Specifically, stromal co-culture assays revealed that combined treatment with dasatinib at 1 nM, and imetelstat at 1 µM or 5 µM significantly inhibited survival and replating of blast crisis (BC) CML progenitors compared with aged bone marrow progenitors (p &amp;lt; 0.001, ANOVA). As a single agent, imetelstat (5 µM) inhibited survival and replating of pre-LSC derived from myelofibrosis compared with normal bone marrow progenitor samples (p &amp;lt; 0.001, ANOVA). In pre-LSC MPN NSG-SGM mouse models established from 4 different MF samples, a significant reduction in proliferation of human CD45+ cells (p &amp;lt; 0.01, t test) was observed in bone marrow and spleen, when compared with vehicle control. Treatment of humanized LSC mouse models, established with 5 different BC CML, with 30 mg/kg of imetelstat, 3 times a week for 4 weeks resulted in a significant reduction in proliferation of malignant progenitors and human CD45+ cells (p &amp;lt; 0.001, ANOVA). As measured by a Flow-FISH assay, abnormal telomere length was reversed by imetelstat treatment compared with mismatch control (p &amp;lt; 0.05, ANOVA). In addition, FACS analysis revealed a significant reduction in activated beta-catenin expression after imetelstat treatment of LSC engrafted mice compared with vehicle control (p &amp;lt; 0.01, ANOVA). Finally, RNA-seq analysis performed on human CD34+ cells from imetelstat treated LSC mouse models revealed a significant reduction in LSC harboring malignant ADAR1-mediated A-to-I editing at doses that spared normal hematopoietic stem cells. CONCLUSIONS Combined WGS and RNA-Seq analyses, lentiviral ADAR1 overexpression, stromal co-culture assays and humanized pre-LSC and LSC mouse model studies reveal that pre-LSC evolution into LSC coincides with both ADAR1 and hTERT activation, which can be prevented with imetelstat. Disclosures Rizo: Geron Corp: Current Employment, Current equity holder in publicly-traded company. Huang:Geron Corp: Current Employment, Current equity holder in publicly-traded company. Jamieson:Forty Seven Inc: Patents &amp; Royalties; Bristol-Myers Squibb: Other.

Let-7b regulates the adriamycin resistance of chronic myelogenous leukemia by targeting AURKB in K562/ADM cells

Chronic myeloid leukemia (CML) is a malignant hematological disease, and drug resistance is often related to poor prognosis. MicroRNAs (miRNA) play a pivotal role in transcriptional regulation, cell development, and chemotherapy resistance. Here, we describe the effect of let-7b on resistant leukemia cells and examine the relevance of let-7b as a biomarker for adriamycin resistance. Results showed that let-7b was downregulated in K562/ADM (KA) cells, and the downregulation of let-7b in K562 and KA cells increased ADM resistance. The inhibition of let-7b subsequently induced the upregulation of AURKB. Finally, results proved that the Pi3k/Akt/Erk pathway was related to AURKB-activated resistance. Our research indicated that the underexpression of let-7b and overexpression of AURKB contributed to the resistance of CML, and its function is partly regulated by the Pi3k/Akt/Erk pathway. Thus, our further understand of its inhibitory effect may promise a new therapeutic strategy to overcome chemotherapeutic resistance in CML.

AT9283 exhibits antiproliferative effect on tyrosine kinase inhibitor‑sensitive and ‑resistant chronic myeloid leukemia cells by inhibition of Aurora A and AuroraB.

Imatinib is the gold standard in the conventional treatment of chronic myeloid leukemia (CML). However, some patients become resistant to imatinib therapy. To overcome this resistance, second‑generation (dasatinib, nilotinib, and bosutinib) and third‑generation (ponatinib) tyrosine kinase inhibitors (TKIs) have been developed and have been shown to be effective against refractory CML. Although these TKIs provide many benefits for patients with CML, advanced patients show resistance even to these TKIs. Therefore, novel therapeutic strategies are urgently needed for the treatment of TKI‑resistant CML patients. AT9283 is a multi‑targeted kinase inhibitor with potent activity against Janus kinase (JAK), Aurora kinases, and Abl. In the present study, we showed that AT9283 significantly decreased the cell viability of both TKI‑sensitive and TKI‑resistant CML cells as determined by trypan blue exclusion assay. In addition, cell cycle analysis, Annexin V assay, and caspase‑3/7 activity assay revealed that AT9283 increased the cell population in the G2/M phase and induced apoptosis. We investigated the molecular mechanisms underlying the decrease in cell viability upon treatment with AT9283 by western blotting. Interestingly, our results showed that AT9283 inhibited the expression of Aurora A, Aurora B, and downstream Histone H3 phosphorylation. In contrast, we observed no changes in the levels of Bcr‑Abl, signal transducer and activator of transcription 3 (STAT3), extracellular signal‑regulated kinase (ERK), and Akt phosphorylation. In addition, we found that AMG900, a selective Aurora A and Aurora B inhibitor, increased the G2/M phase cell population and induced apoptosis via inhibition of Aurora A and Aurora B in both TKI‑sensitive and TKI‑resistant CML cells. Our studies show that Aurora A and Aurora B are promising therapeutic targets for TKI‑sensitive and TKI‑resistant CML, and AT9283 may have potential clinical applications for the treatment of TKI‑resistant CML patients.

Abstract LB-141: Increased epigenetic plasticity and tumor heterogeneity predict resistance to CD19 CAR T cell therapy in pre-treatment pediatric acute lymphoblastic leukemia

Abstract Background: Acute lymphoblastic leukemia (ALL) is the most common childhood cancer with a peak incidence between 3-5 years of age. Despite the improved survival rate of 90% for newly diagnosed children with ALL, the outcome for patients who relapse is poor with a less than 30% survival. CD19 CAR T-cells have shown remarkable response rates between 80-90% in relapsed/refractory disease. Little is known about antigen-independent factors that predict initial resistance to CD19 CAR T-cell therapy. We hypothesized that leukemias that are resistant to CD19 CAR T-cell therapy are distinct from sensitive leukemias and that these differences can be detected prior to therapy. Methods: To interrogate differences in the genomes, transcriptomes, intratumoral heterogeneity, and epigenetic landscapes between resistant and sensitive leukemias, we obtained pre-treatment bone marrow aspirates (BMAs) from patients enrolled in a clinical trial at Seattle Children's Hospital (PLAT-02). Samples were categorized based on patient response and included 7 resistant (2 with matched post-treatment) and 7 sensitive leukemias as matched controls. We performed whole exome sequencing, bulk RNA-seq, array-based methylation, scRNA-seq, CyTOF, ATAC-seq, and H3K27ac ChIP-seq. Additionally, we established CD19 CAR T resistant and sensitive patient derived xenografts (PDXs) of these leukemias, which are currently being used for in vivo modelling of these diseases. Results: We identified mutations and fusions in known epigenetic modifiers, including CREBBP and EP300, in all resistant leukemias. ATAC-seq revealed relatively more accessible chromatin in resistant patients in comparison to sensitive (16,082 vs. 6,070 peaks). The combination of altered epigenetic modifiers and increased chromatin accessibility is suggestive of epigenetic plasticity. Motif analysis of these differentially accessible peaks showed an enrichment for AP-1 binding proteins, particularly JUN and its partners, which provides a mechanism to avoid caspase-induced apoptosis, the downstream effect of cytotoxic T-cell killing. Methylation array and expression data indicate promoter hypermethylation and lowered expression of JUN pathway genes in sensitive leukemias. CyTOF identified a subpopulation with characteristics of both myeloid and lymphoid lineages in one sample that may represent a resistant population of cells selected for under CAR T-cell pressure. Analysis of the marrow microenvironment indicates that patients with sensitive leukemias have increased markers of T-cell activation, suggestive of more robust T-cells capable of rejecting the leukemias. Conclusions: This study establishes one of the most integrative and comprehensive profiling approaches for patient samples. We have shown the association of epigenetic plasticity, JUN pathway upregulation, and decreased T-cell activation with resistance to CD19 CAR T-cell therapy. We are currently validating these results by modelling our cohort's therapy resistance in vivo. Citation Format: Katherine E. Masih, Abdalla Abdelmaksoud, David Milewski, Berkley E. Gryder, Rebecca Gardner, Ashley L. Wilson, Benjamin Z. Stanton, Young K. Song, Chaoyu Wang, Xinyu Wen, Serifat Adebola, Zachary Rae, Adam Cheuk, Justin Lack, Gregoire Altan-Bonnet, Michael Kelly, Jun Wei, Michael Jensen, Rimas J. Orentas. Increased epigenetic plasticity and tumor heterogeneity predict resistance to CD19 CAR T cell therapy in pre-treatment pediatric acute lymphoblastic leukemia [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-141.

Abstract 469: Selective targeting of splicing deregulation in pediatric acute myeloid leukemia stem cells

Abstract While 5 year survival rates for acute lymphoblastic leukemia (ALL) in childhood are 90% (https://www.cancer.org/cancer/leukemia-in-children/detection-diagnosis-staging/survival-rates.html), acute myeloid leukemia survival rates have lagged behind at 65 to 70%. Thus, AML is the leading cause of childhood leukemic mortality and is a heterogeneous disease characterized by diverse mutations many occurring at low frequencies. Whole exome sequencing analyses have not been sufficient to predict relapse in many pediatric patients at least in part because of transcriptomic and epitranscriptomic alterations that drive therapy resistant leukemia stem cell (LSC) propagation. Recent results reported by ourselves and others suggest that LSC in AML harbor unique mRNA splicing profiles characterized by intron retention and exon skipping. In adult AML, whole transcriptome RNA sequencing data revealed global spliceosome disruption that uniquely distinguished LSCs from normal age-matched hematopoietic stem and progenitor cells (Crews et al Cell Stem Cell 2016). However, the role of splicing deregulation in LSC propagation in human pediatric AML had not been clearly elucidated. Recently, we have developed 17S-FD-895, a small molecule compound targeting SF3B1, which modulates mRNA splicing. To date, we have evaluated the effects of this splicing modulator on both self-renewal as well as pro-survival splice variants in CD34+ cells derived from both peripheral blood as well as bone marrow of pediatric AML patients. Splice isoform specific qRT-PCR demonstrated a dose-dependent increase in SF3B1 intron retention following treatment. Furthermore, splicing modulation induced MCL1 exon 2 skipping, producing pro-apoptotic MCL1-S transcripts. Hematopoietic progenitor assays demonstrated a dose-dependent reduction in LSC clonogenicity and self-renewal. In these assays, LSC were found to be sensitive to nanomolar concentrations that spared normal hematopoietic stem and progenitor cells. To further dissect the role of pre-mRNA splicing in pediatric AML, we developed a lentiviral fluorescent splicing reporter that switches from GFP to RFP expression following an exon skipping or intron retention event. In addition, whole transcriptome RNA sequencing (RNA-seq) was performed on FACS purified stem cells (CD34+/CD38-/Lin-) as well as progenitor cells (CD34+/CD38+/Lin-) from both pediatric AML as well as aged-matched normal bone marrow samples and samples were transplanted into immunocompromised mice followed by 17S-FD-895 treatment to assess sensitivity to splicing modulation in vivo. As a result of these studies, we have demonstrated LSC splicing patterns in pediatric AML that may inform novel biomarker identification as well as development of 17S-FD-895 for pediatric AML. Citation Format: Inge Van der Werf, Phoebe Mondala, Raymond Diep, Larisa Balaian, Cayla Mason, Gertjan Kaspers, Jacqueline Cloos, Jim La Clair, Peggy Wentworth, Tom Whisenant, Katie Fisch, Michael Burkart, Catriona Jamieson. Selective targeting of splicing deregulation in pediatric acute myeloid leukemia stem cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 469.

Abstract A11: A comprehensive and integrative omic analysis of multiply relapsed refractory pediatric pre-B cell acute lymphoblastic leukemia predicts response to CD19 CAR T-cell therapy

Abstract Acute lymphoblastic leukemia (ALL) is the most common childhood cancer with a peak incidence at 3-5 years of age. Despite the improved survival rate of 90% for newly diagnosed children with ALL, the outcome for patients with relapsed disease is poor with a less than 30% overall survival. CD19 CAR T-cell therapy has shown remarkable response rates in relapsed/refractory disease. Long-term survival analysis has shown that initial response rates exceed 80%. However, durable response rates at one year are closer to 40%. Little is known about factors predicting durable response to CAR T therapy. We hypothesize that patients with CD19 CAR T-cell resistant ALL have a distinct disease compared to responders to therapy that can be identified in pretreatment leukemia. Utilizing advanced genomic, epigenetic, proteomic, and single-cell (sc) techniques, we characterized patient bone marrow aspirates (BMA) to identify mechanisms of resistance. Patients enrolled in PLAT-02 at Seattle Children’s Hospital were categorized according to the durability of their response to CD19 CAR T therapy. To characterize the molecular and genomic alterations specific to the therapy-resistant ALLs, we performed comprehensive analyses on pre-treatment therapy-resistant and sensitive BMAs using whole-exome sequencing, RNA- BMAs seq, scRNA-seq, sc B cell receptor (BCR)-seq, methylation array, H3K27ac ChIP-seq, ATAC-seq, and CyTOF. Additionally, we developed murine patient-derived xenografts (PDXs) for future studies. Initial mutation analyses revealed 5 hotspot mutations (ABL1, 2 x KRAS, IKZF1, and EP300) and actionable fusion (2 ABL1, 2 ETV6, 2 ETV5, KMT2A). Interestingly, we identified a KMT2A-AFF1 fusion in a sensitive leukemia, which has been demonstrated to predispose patients to CD19 CAR T resistance through lineage switching. Additionally, we identified a novel CREBBP-fusion in leukemias resistant to CD19 CAR T-induced B-cell aplasia. Alterations of CREBBP have previously been associated with ALL that is refractory to conventional therapies. Integrated gene expression and epigenetic analyses are ongoing to identify genes or pathways associated with resistant disease. scRNA- and scBCR-seq data are being analyzed and integrated with CyTOF analyses to detect mixed lineage and gene expression-based heterogeneity that may predict clonal selection by CAR T pressure. Finally, we developed and genetically analyzed murine PDXs for 64% of the patient samples, establishing a valuable resource for future studies and developing novel therapies for resistant leukemias. This study is one of the most integrative and comprehensive genomic profiling approaches to identify the molecular traits of therapy-resistant ALL in patient samples. We hope to identify and develop crucial biomarkers predicting responsiveness to CAR T-cell therapy. Citation Format: Katherine E. Masih, Rebecca Gardner, Berkley E. Gryder, Abdalla Abdelmaksoud, Ashley Wilson, Serifat Adebola, Benjamin Z. Stanton, Young K. Song, Justin Lack, Chaoyu Wang, Xinyu Wen, Zachary Rae, Adam Cheuk, Gregoire Altan-Bonnet, Michael Kelly, Jun S. Wei, Michael C. Jensen, Rimas J. Orentas, Javed Khan. A comprehensive and integrative omic analysis of multiply relapsed refractory pediatric pre-B cell acute lymphoblastic leukemia predicts response to CD19 CAR T-cell therapy [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr A11.

A novel HDAC inhibitor chidamide combined with imatinib synergistically targets tyrosine kinase inhibitor resistant chronic myeloid leukemia cells

Chidamide is a novel selective histone deacetylase inhibitor (HDACi) with promising activity in hematological malignancies, but its role in chronic myeloid leukemia (CML) was marginally addressed. In this study, we firstly demonstrated that chidamide alone inhibited CML cells proliferation, induced apoptosis and cell cycle arrest. Further, chidamide combined with imatinib (IM) induced synergistic lethality in CML cell line KBM5, as well as IM-resistant CML cells KBM5T315I, associated with a marked reduction of Bcr-Abl kinase activity and acetyl-histone H3 expression. The combination treatment markedly inhibited constitutive activity of β-catenin signaling in IM-resistant cells and abolished the protective effects of mesenchymal stromal cells (MSCs) to CML cells. In addition, the co-treatment significantly reduced Bcr-Abl and β-catenin transcript levels and induced apoptosis of primary CD34+ stem/progenitor cells derived from blast crisis (BC)-CML patients, but exhibited minimal toxicity to normal CD34+ progenitors. Collectively, our data show that combination of chidamide and imatinib synergistically targets tyrosine kinase inhibitor (TKI) -resistant BC-CML cells via inhibition of Bcr-Abl and β-catenin signaling, suggesting that this combination has the potential for treating TKI-resistant CML and improving clinical outcomes of BC-CML patients.

Cytotoxic Activity of Germacrane-Type Sesquiterpene Lactones from Dimerostemma aspilioides.

The need for effective candidates as cytotoxic drugs that at the same time challenge cancer multidrug resistance encouraged a search for these in plants of central Argentina. Bioassay-guided fractionation of the cytotoxic extract from Dimerostemma aspilioides led to the isolation of the germacranolide tomenphantin A (1), along with three new analogues (2-4). These efficiently inhibited the proliferation of the leukemia cell lines K562 and CCRF-CEM and their resistant variants, Lucena 1 and CEM/ADR5000, respectively, with IC50 values ranging from 0.40 to 7.7 μM. The structures and relative configurations of compounds 1-4 were elucidated by analysis of the spectroscopic data, in particular NMR spectroscopy. The most active among these was compound 1 (IC50 = 0.40-5.1 μM), and, therefore, this was selected as a model for a mechanistic study, which revealed that its antiproliferative effect was mediated by cell cycle arrest in the G2/M phase followed by apoptosis. The activity of compound 1 was selective, given the absence of cytotoxicity toward peripheral blood mononuclear cells. The results show the potential of these compounds, and in particular of compound 1, as leads for the development of drug candidates to fight sensitive and resistant leukemia cells.

N-alkylisatin-based microtubule destabilizers bind to the colchicine site on tubulin and retain efficacy in drug resistant acute lymphoblastic leukemia cell lines with less in vitro neurotoxicity

BackgroundDrug resistance and chemotherapy-induced peripheral neuropathy continue to be significant problems in the successful treatment of acute lymphoblastic leukemia (ALL). 5,7-Dibromo-N-alkylisatins, a class of potent microtubule destabilizers, are a promising alternative to traditionally used antimitotics with previous demonstrated efficacy against solid tumours in vivo and ability to overcome P-glycoprotein (P-gp) mediated drug resistance in lymphoma and sarcoma cell lines in vitro. In this study, three di-brominated N-alkylisatins were assessed for their ability to retain potency in vincristine (VCR) and 2-methoxyestradiol (2ME2) resistant ALL cell lines. For the first time, in vitro neurotoxicity was also investigated in order to establish their suitability as candidate drugs for future use in ALL treatment.MethodsVincristine resistant (CEM-VCR R) and 2-methoxyestradiol resistant (CEM/2ME2-28.8R) ALL cell lines were used to investigate the ability of N-alkylisatins to overcome chemoresistance. Interaction of N-alkylisatins with tubulin at the the colchicine-binding site was studied by competitive assay using the fluorescent colchicine analogue MTC. Human neuroblastoma SH-SY5Y cells differentiated into a morphological and functional dopaminergic-like neurotransmitter phenotype were used for neurotoxicity and neurofunctional assays. Two-way ANOVA followed by a Tukey’s post hoc test or a two-tailed paired t test was used to determine statistical significance.ResultsCEM-VCR R and CEM/2ME2-28.8R cells displayed resistance indices of > 100 to VCR and 2-ME2, respectively. CEM-VCR R cells additionally displayed a multi-drug resistant phenotype with significant cross resistance to vinblastine, 2ME2, colchicine and paclitaxel consistent with P-gp overexpression. Despite differences in resistance mechanisms observed between the two cell lines, the N-alkylisatins displayed bioequivalent dose-dependent cytotoxicity to that of the parental control cell line. The N-alkylisatins proved to be significantly less neurotoxic towards differentiated SH-SY5Y cells than VCR and vinblastine, evidenced by increased neurite length and number of neurite branch points. Neuronal cells treated with 5,7-dibromo-N-(p-hydroxymethylbenzyl)isatin showed significantly higher voltage-gated sodium channel function than those treated with Vinca alkaloids, strongly supportive of continued action potential firing.ConclusionsThe N-alkylisatins are able to retain cytotoxicity towards ALL cell lines with functionally distinct drug resistance mechanisms and show potential for reduced neurotoxicity. As such they pose as promising candidates for future implementation into anticancer regimes for ALL. Further in vivo studies are therefore warranted.