Central nervous system (CNS) involvement is an extremely rare extramedullary multiple myeloma (MM) manifestation, diagnosed in less than 1% of patients. It is considered an ultimate high-risk feature, associated with unfavorable cytogenetics, and, even with intense treatment applied, survival is short, reaching less than 12 months in most cases. In June 2017 an 81 years old male with a κ light chain MM was referred to our institution for an isolated CNS MM relapse. His cerebrospinal fluid (CSF) demonstrated a high load of clonal plasma cells, however, the patient's bone marrow infiltration was very little with a percentage of plasma cells less than 5%. Imaging, including gold standard MRI and experimental 11C-methionine PET scan, was performed, and high metabolic activity was detected supra- and infratentorially as well as in the right femur and the clivus. Following CD138+ cell purification we analyzed the specimen with M3P (v3.0) a disease specific in-house customized, next generation targeted sequencing panel for MM (Ion torrent platform). This includes most commonly mutated MM genes, actionable drug targets and drug resistance associated genes. The average sequencing depth increased 700X and spatial MM heterogeneity was detected, as the CFS cells harbored a clonal BRAFV600E mutation, absent in the bone marrow. Initial intrathecal and systemic chemotherapy with Cytarabine and Thiotepa was intolerable, thus the patient underwent a combined target inhibition with Dabrafenib/Trametinib, well known specific BRAF and a MEK 1/2 inhibitors. The patient displayed a rapid complete response (Figure. 1A), however, disease relapse occurred after three months of therapy. We obtained a sequential CFS sample and Whole Exome Sequencing (Illumina platform) was applied to pre and post therapy CFS sampling. Exome sequencing of the two time points performed an average sequencing depth of 115X; a total number of 97 non-silent coding variants (missense, nonsense, indels, splice) with an allele frequency higher than 5% were detected. In detail, 19 point mutations were acquired at relapse, including a subclonal missense mutation in CIC (p.A984P, VRF 17%), recently identified as a candidate gene contributing to MEK/BRAF resistance development. Next, we established a CIC knock-down model electroporating a specific anti-CIC siRNA into U266 MM cell line. We cultured the silenced and not-silenced cells with Trametinib and Dabrafenib, either as single agents, or in combination. As expected, we observed resistance induction to the combination of the two drugs (Row Factor 85.94%; P<0.0001, Two-way ANOVA) suggesting a critical role for this patient derived mutation for his MEK/BRAF resistance development (Figure 1C, D). In order to better clarify the landscape pathway related to CIC we analyzed expression data from 647 patients enrolled in the MMRF CoMMpass trial. Remarkably, we found a significant down-regulation of ERF and ETV6 (t-test -9.95, -9.93, P <0.001, respectively), two well characterized tumor suppressor genes correlated with the re-activation of the RAS downstream pathway (Figure 1B). This is the first report giving evidence for a potential role of point mutations in CIC as a resistance mechanism to targeted MEK/BRAF inhibition in BRAF mutated MM. The performed pathway analysis significantly extends the insights of the resistance mechanisms highlighted. Our results foster a statistically powered study to corroborate the clinical relevance. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.
Introduction Acute Myeloid Leukemia (AML) is a genetically heterogeneous disease characterized by clonal expansion of immature myeloid progenitor cells in the bone marrow (BM). Mutations of the FMS-like tyrosine kinase 3 (FLT3) gene occur in approximately 30% of AML cases, with Internal Tandem Duplications (ITD) being the most common type of mutation. Several FLT3 specific inhibitors (TKI) have been developed such as quizartinib, crenolanib and midostaurin (recently approved for clinical use). Nevertheless FLT3-ITD is associated with unfavorable prognosis and patients develop drug resistance with the underlying mechanisms remaining largely unexplained. Recently, changes within the actin cytoskeleton were associated with drug resistance development in various cancers. FLT3-ITD mutations are associated with RAC1 activation. RAC1 belongs to the family of RHO GTPases and enhances the actin polymerization by inducing the expression of N-WASP or WAVE2 and ARP2/3 complex. Therefore, we investigated actin cytoskeleton rearrangements through RAC1 activation as a potential mechanism contributing to Midostaurin resistance in AML. Material and methods First, we developed two Midostaurin resistant AML cell lines (MID-RES, MV4-11 and MOLM-13). Single cell measurements of Cell Stiffnes, cell adhesion forces between tumor and HS5 stroma cells and Actin filaments were performed by Atomic Force Microscopy (FluidFM®) and SIM microscopy, respectively. RAC1 activation was measured by RAC1 activation kit provided by Cytoskeleton. FLT3 surface and intracellular expression was measured by Flow cytometry and western blot, respectively. Cell death was analyzed by Annexin/PI staining in flow cytometry. Results The MID-RES cell lines MV4-11/MOLM-13 showed higher FLT3 surface and intracellular expression compared to their MID sensitive parental cells. In line with our expectations, we observed RAC1 activation, as well as an up-regulation of actin polymerization positive regulators such as N-WASP, WAVE2, PFN1 and ARP2/3 complex and the inhibition of actin polymerization negative regulator P-ser3 CFL1 in MID-RES cells. FLT3 receptor knock down by siRNAs reversed the MID resistance and reduced RAC1 activation and actin polymerization inducers expression. Likewise, bioinformatic analysis from publicly available microarray expression data (E-MTAB-3444), confirmed positive correlation between actin polymerization inducers and FLT3 signaling expression in 178 FLT3-ITD (r=0,67) and 461 FLT3 WT(r= 0,57) de novoAML patients. RAC1 induced Actin polymerization positively correlates with actin filaments growth and cell stiffness, which was observed in our MID-RES cells, higher load of actin filaments and increased cell stiffness. The combination between RAC1 specific inhibitor, EHT1864 and Midostaurin synergistically induces cell death in MID-RES cells by arresting cell cycle in G0/G1 phase and activating apoptosis. Beside, this combination reduced the adhesion forces to stroma cells, decreased the expression of PFN1/N-WASP/ARP2 and consequently reduced drastically the number of actin filaments and cell stiffness in MID-RES cells. EHT1864 and Midostaurin (alone and in combination) were not toxic in PBMCs obtained from healthy donors. Interestingly, this combination increase >45 % cell death in cells obtained from refractory FLT3-mutated AML patient (this patient was relapsed (≥ 50% residual blasts in the bone marrow)under Chemotherapy+Midostaurin combination).The specific knock down of PFN1/N-WASP/ARP2 with siRNAs equally reversed the resistance to Midostaurin. Of note, RAC1 regulates the anti-apoptotic BCL2. Indeed, EHT1864 in combination with Midostaurin reduced anti-apoptotic family BCL2/MCL1 expression and increases the pro-apoptotic proteins BAX/PUMA. As expected, our MID-RES cells showed higher sensitivity to BCL2 inhibitor Venetoclax, than their parental cells. The combinations EHT1864+venetoclax, venetoclax+midostaurin and venetoclax+Midostaurin+EHT1864 synergistically induced cell death in MID-RES cells. Conclusion Actin polymerization inducers N-WASP, ARP2/3 complex and PFN1 may provide a valuable approach to overcome Midostaurin resistance in AML. Our data further suggest that the addition of BCL2 inhibition through EHT1864 and venetoclax could represent an interesting strategy to potentiate the activity of Midostaurin in FLT3 mutated AML. Disclosures Duell: Regeneron Pharmaceuticals, Inc.: Research Funding. Rosenwald:MorphoSys: Consultancy.
Acute Myeloid Leukemia (AML) is a genetically heterogenous disease characterized by clonal expansion of immature myeloid progenitors cells in the bone marrow (BM). Despite this genetic heterogeneity, AML patients share Leukemia associated oncogenes such as NF-E2-related factor 2 (Nrf2) (Rushworth SA et al.). NRF2 is a transcription factor that activates genes with antioxidant response elements (ARE)-containing promoters and protects cancer cells from apoptosis. Inhibition of NRF2 or antioxidant defense increases the level of Radical Oxygen Species (ROS), leading to tumor supression (Chio IIC et al.). Recently, the E3 Ubiquitin-Protein Ligase HACE1, a tumor suppressor in solid tumors, was demonstrated to promote the expression of NRF2 in Huntigton disease (Rotblat B et al.). Thus, we hypothesized a role for HACE1 as an oncogenic factor acting through NRF2 activation in myeloid malignancies and provide first data supporting the HACE1-NRF2 axis to be a novel target in acute myeloid leukemias. Material and methods The mRNA expression data from AML patients (296 samples) vs normal Hematopoietic Stem Cells (HSC) (6 samples) were exported from the bloodSpot database. HACE1 mRNA and protein expression was measured by q-RT-PCR and western blot in 12 commercially available Myeloid Malignancies cell lines. The HACE1 inducible knock down (KD) was carried out by Sleeping Beauty Transposon system in U937 and NOMO-1 cell lines. The cell viability was analyzed by Cell Titer Glo Luminescent assay. Apoptosis was measured by Annexin V (AV)/Propidium Iodide (PI) assay. Results and discussion HACE1 mRNA is downregulated in AML patients compared to HSC (***p<0.001, Bloodspot database). However mRNA and HACE1 protein expression do not correlate in AML cell lines, suggesting post translational modifications. High HACE1 protein expression was observed in most AML cell lines. HACE1 KD reduced drastically the cell viability of U937 cells through caspase activation and NRF2 degradation. However, no effect on cell viability was observed in NOMO-1 cells. Recently, non-programmed cell death necroptosis induction has been described by TNFR1 activation in HACE1 knock out Mouse Embrionic Fibroblast cells (Tortola L et al.). In line with this study, we observed that TNF induces strong cell death in HACE1 KD NOMO-1 cells within 48 hours. In addition HACE1 KD promotes autophagy through p62 degradation (late autophagy marker) in U937 cells. Autophagy has recently been described to contribute to the differentiation and death of AML cells, and to the promotion of immunostimulatory signals activating immune responses against cancer cells (Chen L et al.; Pietrocola F et al.). Thus HACE1 might be a potential target to induce autophagy, providing a novel therapeutical target in the treatment of myeloid malignancies. Finally, HACE1 KD in our hands promoted sensitization of U937 and NOMO-1 cells to cytatarabine, the backbone therapy in AML patients. This treatment promotes HACE1 protein expression at 24 and 48 hours in NOMO-1 cells, which may explain the better response rates of HACE1 KD cells to cytarabine. Taken together, we provide first evidence of HACE1 being a novel oncogene in AML and that the HACE1-NRF2 axis is a promising target in the treatment of Acute Myeloid Leukemias. Disclosures Haferlach: MLL Munich Leukemia Laboratory: Employment, Equity Ownership.
Secondary Acute Myeloid Leukemia (sAML) accounts for 10-30% of all AML. It arises from a preexisting clonal disorder of hematopoiesis, such as myelodysplastic syndromes (MDS) or chronic myeloproliferative neoplasia (cMPN) in most cases (60-70%) or from exposure to a leukemogenic agent e.g. chemotherapy. sAML is generally considered to be of unfavorable prognosis, as treatment sensitivity is reduced, compared to de novo AML (dnAML) and overall survival is shortened. The incidence of AML associated NRAS are similar between sAML and dnAML (10 to 15%, Jelena D. Milosevic et al.). Prognostic impact of such mutations have been controversially discussed, but have been linked to favorable response to high dose cytarabine treatment in dnAML patients (Andreas Neubauer et al.), thus providing the first example of an oncogenic mutation impacting drug response in dnAML. This effect, however, has not yet been shown in sAML, therefore the aim of this work is to study the role of mutated NRAS in the response to chemotherapy and the hypomethylating agent (HMA) 5-azacitidine in sAML. We utilized two sAML cell lines SET-2 and HEL (both NRAS wildtype) in which we stably introduced the NRAS WT and the known activating hotspot mutation NRAS G12D using the sleeping Beauty technology. The dose-response assays of conventional chemotherapy and 5-azacitidine were carried out in the parental cell lines (SET-2/HEL) compared to NRAS WT (SET-2 NRAS WT/HEL NRAS WT) and NRAS G12D (SET-2 NRAS G12D/HEL NRAS G12D). In contrast to our expectations, both NRAS G12D mutation harboring cell lines, SET-2 and HEL developed resistance to cytarabine, idarubicin and 5-azacytidine, whereas the ones with wildtype NRAS remained susceptible to the drugs. To reverse the resistance we tested the MEK inhibitors Binimetinib and Trametinib in our SET-2 NRAS G12D cell line model according to recent reports about preclinical efficacy of MEK inhibition in NRAS mutant dnAML cells (Michael R. Burgess et al.). And in fact, single agent Binetimib and Trametinib treatments reduced cell viability by 20% at 48 hours. Strikingly, in combination with 5-azacitidine, Binimetinib and Trametinib treatments led to a viability reduction by 90%. Next we induced necroptosis in our NRAS mutant cell line models through the combination of Birinapant (SMAC mimetics) and Emricasan (Inhibitor of Caspase 8), as recently described by Brumatti et al. and were, in addition, able to reduce the cell viability by 60 %. In summary, we provide first evidence, that in contrast to dnAML, activating NRAS mutations may promote resistance to conventional chemotherapy and 5-azacitidine in sAML cell lines. Furthermore we were able to demonstrate, that the combination of MEK inhibitors (Binimetinib and Trametinib) and 5-azacitidine as well as the induction of necroptosis such as the combination birinapant and emricasan, may provide a potential strategy to overcome the resistance. Disclosures Haferlach: MLL Munich Leukemia Laboratory: Employment, Equity Ownership.
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