Alterations of RAS
 signalling in Chinese multiple myeloma patients: absent BRAF
 and rare RAS
 mutations, but frequent inactivation of RASSF1A
 by transcriptional silencing or expression of a non-functional variant transcript

Abstract Overexpression of fibroblast growth factor receptor 3 (FGFR3) is a hallmark of t(4;14) multiple myeloma (MM). To dissect the mechanism of FGFR3 oncogenesis in MM, we used 3 FGFR selective kinase inhibitors—CHIR258, PD173074, and SU5402—and FGFR3-specific siRNA to modulate FGFR3 activity. Conversely, the ligand FGF was used to stimulate FGFR3 function in human MM cells. The transcriptional response to FGFR3 modification was recorded, and gene expression changes common to all 5 modifiers were documented. Ten genes were commonly regulated. Macrophage inflammatory protein-1 alpha (MIP-1α) was the single most differentially altered gene. MIP-1 α promoter function, gene expression, and protein secretion were each down-regulated following inhibition of FGFR3 signaling. Down-regulation of MIP-1 α was not, however, observed following FGFR3 inhibition in MM cells with RAS mutations implicating RAS-MAPK in MIP-1 α regulation. As confirmation, inhibition of ERK1 also down-regulated MIP-1 α in FGFR3 inhibitor-resistant cells harboring RAS mutations. MIP-1 α is implicated in the survival and proliferation of MM cells and the pathogenesis of MM bone disease. Our observation is the first to directly link an initiating IgH translocation not only to MM-cell growth and survival but also to the disease-associated bone disease.

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Frequent functional activation of RAS signalling not explained by RAS/RAF mutations in relapsed/refractory multiple myeloma

Scientific Reports ◽

10.1038/s41598-018-31820-9 ◽

2018 ◽

Vol 8 (1) ◽

Cited By ~ 7

Author(s):

Kwan Yeung Wong ◽

Qiumei Yao ◽

Ling-Qing Yuan ◽

Zhenhai Li ◽

Edmond Shiu Kwan Ma ◽

...

Keyword(s):

Multiple Myeloma ◽

Refractory Multiple Myeloma ◽

Ras Signalling

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RAS mutations – for better or for worse in multiple myeloma?

Leukemia & Lymphoma ◽

10.3109/10428194.2015.1065984 ◽

2015 ◽

Vol 57 (1) ◽

pp. 8-9 ◽

Cited By ~ 2

Author(s):

Ellen Leich ◽

Torsten Steinbrunn

Keyword(s):

Multiple Myeloma ◽

Ras Mutations

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Targeting GCK pathway: a novel and selective therapeutic strategy against RAS mutated multiple myeloma

Blood ◽

10.1182/blood.2020006334 ◽

2020 ◽

Author(s):

Shirong Li ◽

Jing Fu ◽

Jun Yang ◽

Huihui Ma ◽

Divaya Bhutani ◽

...

Keyword(s):

Multiple Myeloma ◽

Cell Proliferation ◽

Growth Inhibition ◽

Alternative Therapy ◽

Braf Mutations ◽

Ras Mutation ◽

Ras Mutations ◽

Cell Proliferation Inhibition

In multiple myeloma (MM), frequent mutations of NRAS, KRAS, or BRAF are found in up to 50% of newly diagnosed patients. The majority of the NRAS, KRAS, and BRAF mutations occur in hotspots causing constitutive activation of the corresponding proteins. Thus targeting RAS mutation in MM will increase therapeutic efficiency and potentially overcome drug-resistance. We identified Germinal Center Kinase (GCK) as a novel therapeutic target in MM with RAS mutation. GCK knockdown in MM cells demonstrated in vitro and in vivo that silencing of GCK induces MM cell growth inhibition, associated with blocked MKK4/7-JNK phosphorylation and impaired degradation of IKZF1/3, BCL-6, and c-MYC. These effects were rescued by overexpression of an shRNA-resistant GCK, thereby excluding the potential off-target effects of GCK knockdown. In contrast, overexpression of shRNA-resistant GCK kinase-dead mutant (K45A) inhibited MM cell proliferation and failed to rescue the effects of GCK knockdown on MM growth inhibition, indicating that GCK kinase activity is critical for regulating MM cell proliferation and survival. Importantly, the higher sensitivity to GCK knockdown in RASMut cells suggests that targeting GCK is effective in multiple myeloma which harbors RAS mutations. In accordance with the effects of GCK knockdown, the GCK inhibitor TL4-12 dose-dependently downregulated IKZF1 and BCL-6 and led to MM cell proliferation inhibition accompanied by induction of apoptosis. Hereby our data identify GCK as a novel target in RASMut MM cells, providing a rationale to treat RAS mutations in MM. Furthermore, GCK inhibitors might represent an alternative therapy to overcome IMiD-resistance in MM.

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Macrophage Inflammatory Protein (MIP)-1alpha (CCL3) Is a Downstream Target of FGFR3 and RAS Signaling in Multiple Myeloma.

Blood ◽

10.1182/blood.v106.11.251.251 ◽

2005 ◽

Vol 106 (11) ◽

pp. 251-251

Author(s):

Esther Masih-Khan ◽

Suzanne Trudel ◽

ZhiHua Li ◽

Vincent Nadeem ◽

Ellen Wei ◽

...

Keyword(s):

Multiple Myeloma ◽

Map Kinase ◽

Kinase Inhibitors ◽

Gene List ◽

Ras Signaling ◽

Down Regulation ◽

Ras Mutations ◽

Downstream Target ◽

Inflammatory Protein ◽

Macrophage Inflammatory Protein

Abstract The identification of the t(4;14) translocation in Multiple Myeloma (MM) provided the first indication that fibroblast growth factor 3 (FGFR3) could act as an oncogene. Several studies have assessed the potential of FGFR3 as a drug target and have shown that inhibition of FGFRs using tyrosine kinase inhibitors induces cell death of FGFR3 expressing MM cells. To clarify the signaling pathways implicated in FGFR3 dependent myeloma, we assessed gene expression changes associated with treatment of MM cell lines with three known small molecule FGFR inhibitors, PD173074, SU5402 and a third novel compound using Human U133_Plus2 arrays. In addition to describing the unique pharmacogenomic signals of each drug in myeloma, 845 differentially expressed genes, common to all three FGFR3 inhibitors, were identified. Cell cycle, DNA replication and ATP binding proteins were the most common (47%) functional classes of genes effected. Further validation and refinement of this gene list was conducted using FGFR3 siRNA inhibition and FGF ligand induction. 120 genes were altered between FGFR3 RNAi and scrambled control and 892 were induced by FGF ligand. Ten genes were commonly identified as significantly regulated by all 4 FGFR3 inhibitors and induced in the opposing direction by ligand. Of these macrophage inflammatory protein (MIP)-1alpha, and dual specific phosphatase 6 (DUSP6) were positively regulative while ANXA9, CR2, AL531683, ZNF589, AW274468, FRMD3, LTB and WDR42A were negatively regulated by FGFR3 signaling. Validating our findings, a feedback loop between FGFR/ERK/MAP kinase activation and DUSP6 has previously been described in the literature. MIP-1alpha however was the most significantly altered (12 fold) on array, and MIP-1alpha regulation in response to FGFR3 pathway stimulation was confirmed by flow cytometry and Western blot. Mining of publicly available array datasets on 174 MM patients significantly associated MIP1-alpha with FGFR3 expression (p=0.01). Of note down-regulation of MIP-1alpha was not observed following FGFR3 inhibition in MM cells with RAS mutations. Based on these, and previous findings, we hypothesized that MIP-1alpha was regulated by the FGFR3/ERK/MAP kinase pathway. Indeed, inhibition of ERK in FGFR inhibitor resistant cells with RAS mutations also led to down regulation of MIP-1alpha. Because of the recognized role of MIP1-alpha (CCL3) in survival and proliferation of MM cells and in MM bone disease, our observations raise the possibility that pharmacological inhibition of MIP-1 alpha may hold therapeutic promise in t(4;14) MM and may serve as a biomarker for successful FGFR3 or RAS signaling inhibition.

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Genetic Mutational Panel Analyses of Extramedullary Relapses in Multiple Myeloma; No Gain of RAS Mutations

Blood ◽

10.1182/blood.v126.23.4174.4174 ◽

2015 ◽

Vol 126 (23) ◽

pp. 4174-4174

Author(s):

Monique C. Minnema ◽

Sanne de Haart ◽

Tuna Mutis ◽

Marco Koudijs ◽

Marja van Blokland ◽

...

Keyword(s):

Multiple Myeloma ◽

Soft Tissue ◽

Molecular Mechanisms ◽

P53 Protein ◽

Diagnostic Techniques ◽

Braf V600e ◽

Biopsy Material ◽

Tp53 Mutations ◽

Ras Mutations ◽

The Difference

Abstract Soft tissue extramedullary (EM) disease relapse in Multiple Myeloma (MM) is considered to be a late and aggressive form of the disease with a very poor prognosis. The molecular mechanisms underlying EM disease are unknown but RAS mutations have been implied. To gain further insight in RAS and other mutations in EM relapse, we retrospectively selected MM patients from the hospital database with a relapse EM biopsy from 2000-2015. EM relapse was defined as having a previous diagnosis of MM and a soft tissue EM relapse, with or without bone marrow (BM) involvement. De study was approved by the Scientific Advisory Board Biobanking of the UMC Utrecht. In total, 13 EM samples were retrieved and in 11 of them a BM biopsy was also available at diagnosis (BM-d) and/or at relapse (BM-r). DNA was retrieved from the biopsy material and used in a targeted panel of 50 tumor suppressor and oncogenes using next generation sequencing (NGS). NGS was performed on the IonTorrent PGM using AmpliSeq Cancer Hotspot V2 Panel. This panel primarily contains amplicons to detect currently known, actionable, mutations and amplifications in solid tumors in the following genes ABL1, AKT1, ALK, APC, ATM, BRAF, CDH1, CDKN2A, CSF1R, CTNNB1, EGFR, ERBB2, ERBB4, EZH2, FBXW7, FGFR1, FGFR2, FGFR3, FLT3, GNA11, GNAS, GNAQ, HNF1A, HRAS, IDH1, IDH2, JAK2, JAK3, KDR, KIT, KRAS, MET, MLH1, MPL, NOTCH1, NPM1, NRAS, PDGFRA, PIK3CA, PTEN, PTPN11, RB1, RET, SMAD4, SMARCB1, SMO, SRC, STK11, TP53 and VHL. In addition, immunohistochemistry (IHC) for p53 protein expression was performed on EM biopsies. The EM biopsies were taken from the lymph node (2), pleura (2), skin (7), orbita (1), palate (1) and pancreas (1). The NGS results are presented in Table 1. In total 9 out of 15 BM biopsies yielded results and 10 out of 14 EM biopsies. In 6 patients paired analysis could be performed on both the BM and the EM relapse (EM-r). The most frequent detected mutations were in NRAS (Q61R/K/H) and KRAS (Q61H/L and G13C). These mutations were detected in 5 patients in their diagnostic BM biopsy and in 6 patients in a relapse biopsy. The RAS mutations were mutual exclusive. In total 9 out 13 patients (69%) had a RAS mutation in the diagnostic BM and/or the EM relapse sample. Frequency of RAS mutations in this cohort is higher than previously reported frequencies of 23-44% in newly diagnosed and relapsed MM patients. This suggests an over-representation of these mutations in MM patients with EM relapse, but also the small cohort size or other diagnostic techniques may explain the difference. Remarkable is the lack of difference in frequency of RAS mutations found at time of diagnosis and at time of EM relapse, contradicting the notion that the mutation is acquired during the disease progression from intramedullary to EM disease. TP53 mutations or frameshifts were found in 3 patients (nr 1,18,19). These patients all showed diffuse and strong nuclear expression of the p53 protein on IHC, also indicative for a TP53 mutation. Patient 9 and 10 had p53 protein overexpression in the EM relapse whereas their BM samples had normal and overexpression of TP53, respectively. This is consistent with the general understanding that TP53 mutations are rarely present at time of diagnosis but are more frequent in advanced disease and EM disease. In conclusion, we demonstrate the feasibility of performing NGS on formalin and decalcified BM biopsy material of MM patients. Patients with an EM relapse have a high frequency of 69% of RAS mutations, in most of them already present at diagnosis. The frequency of TP53 mutations is less and mostly detected in relapsed samples. No clear mutations were associated with the progression of intramedullary to EM disease. Table 1. Patient Sample NGS Results Allele frequency Estimated in % 1 BM-d No mutations BM-r NRAS Q61K TP53 R248Q TP53 S241F 34 35 35 EM-r NRAS Q61K 46 3 BM-d NRAS Q61K 16 EM-r Not qualified 4 BM-d KRAS Q61H 38 EM-r Not qualified 6 BM-d Not qualified EM-r NRAS Q61H 52 9 BM-d KIT C840Y 47 BM-r KIT C840Y/C844Y 45 EM-r KIT C840Y/C844YKRAS Q61L 48 62 10 BM-d NRAS Q61R 36 EM-r NRAS Q61R 63 EM-r NRAS Q61R 42 11 BM-d ATM L2877F APC E1317Q 19 34 EM-r ATM L2877FAPC E1317Q 49 63 12 BM-d Not qualified EM-r BRAF V600E 59 17 EM-d No mutations EM-r No mutations 18 BM-d Not qualified EM-r KRAS G13C TP53 frameshift 44 82 19 BM-d Not qualified EM-r TP53 V197L 90 20 BM-d KRAS Q61H 45 EM-r Not qualified 23 BM-r KRAS Q61H 48 EM-r KRAS Q61H 96 Disclosures Minnema: Amgen: Consultancy; Jansen Cilag: Consultancy; Celgene: Consultancy.

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RAS mutations are uncommon in multiple myeloma and other monoclonal gammopathies

International Journal of Oncology ◽

10.3892/ijo.27.4.1023 ◽

2005 ◽

Author(s):

Paloma Martín ◽

Almudena Santón ◽

Mónica García-Cosío ◽

Carmen Bellas

Keyword(s):

Multiple Myeloma ◽

Ras Mutations ◽

Monoclonal Gammopathies

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Prenylation Inhibitors Block IL-6 and IGF-1 Dependent Activation of RAS Signaling in Multiple Myeloma Cells.

Blood ◽

10.1182/blood.v106.11.3373.3373 ◽

2005 ◽

Vol 106 (11) ◽

pp. 3373-3373

Author(s):

Christoph W.M. Reuter ◽

Michael A. Morgan ◽

Dietrich Peest ◽

Arnold Ganser

Keyword(s):

Multiple Myeloma ◽

Cell Proliferation ◽

Western Blotting ◽

Plasma Cell ◽

Hematologic Malignancy ◽

Single Agent ◽

Ras Signaling ◽

Farnesyltransferase Inhibitors ◽

Ras Mutations ◽

Cd34 Cells

Abstract Multiple myeloma (MM) is a fatal hematologic malignancy associated with disruption of RAS-to MAP kinase (ERK) signaling. The IL-6 cytokine family and growth factors such as IGF-1 have been demonstrated to promote malignant plasma cell proliferation through stimulation of ERK and PI-3 kinase/AKT signaling. Prenylation inhibitors such as farnesyltransferase inhibitors (FTIs), geranylgeranyl transferase inhibitors (GGTIs) and the competitive HMG-CoA reductase inhibitor lovastatin have been shown to block RAS post-translational modification and disrupt RAS signaling. To assess efficacy of prenylation inhibitors (e.g. FTI L-744,832, GGTI-2147 and lovastatin) to attenuate MM cell responses to IL-6 and IGF-1, inhibitor-treated cells were analyzed by proliferation/viability assays (MTS) and Western blotting to determine phosphorylated MEK-1/2 and ERK-1/2. FTI L-744,832 was found to inhibit growth of MM cells cultured with IL-6 or IGF-1 even more potently than in cytokine/growth factor-free medium (IC50s 1.3 μM, 1.8 μM and 4.2 μM, respectively). IL-6 moderately protected MM cells from inhibitory effects of GGTI-2147, while IGF-1 had no effect (IC50s 1.1 μM and 0.5 μM vs. 0.5 μM, respectively). IL-6 and IGF-1 protected MM cells from lovastatin-induced growth inhibition (IC50s 4.7 μM and 5.0 μM vs. 1.4 μM, respectively). Furthermore, co-treating MM cells with FTI L-744,832 and GGTI-2147 or lovastatin synergistically inhibited proliferation of MM cells regardless of the presence or absence of IL-6 or IGF-1. Western blotting demonstrated that FTI/GGTI or FTI/lovastatin co-treatment more completely blocked activation of MEK-1/2 and ERK-1/2 in NCI-H929 cells than treatment with any of the compounds alone. Co-treatment also elicited greater inhibition of IL-6 and IGF-1 induced MEK-1/2 and ERK-1/2 activation in NCI-H929 cells. IL-6, IGF-1 and prenylation inhibitors had no effects on AKT phosphorylation status in NCI-H929 cells. To evaluate the clinical relevance of these observations, primary MM cells were obtained from bone marrow aspirates (n=6) or from peripheral blood of a patient with plasma cell leukemia/MM (PCL/MM). Primary MM cells were isolated by magnetic cell sorting using CD138-coupled microbeads, titrated with prenylation inhibitors in the presence of IL-6 and synergy was calculated using the CalcSyn program. Activating RAS mutations (4 K-RAS, 1 N-RAS) were found in 4/7 (57%) MM patient samples, with one sample harboring both K- and N-RAS mutations. FTI L-744,832 elicited anti-myeloma effects only at concentrations much higher than those found to inhibit healthy donor CD34+ cells (IC50’s 51–396 μM vs. 8.2μM) or MM cell lines (1.1–23.8μM) and thus may be ineffective or cause non-specific toxicity when used as a single agent. However, IC50’s calculated for GGTI-2147 and lovastatin were generally higher for CD34+ cells as compared to primary MM cells, supporting specificity of these compounds. Furthermore, combination of FTI with GGTI or lovastatin synergistically inhibited primary MM cell proliferation (IC50’s 0.6–23.1 μM). Our results support that inhibition of RAS down-stream signaling is a major mechanism through which FTI/GGTI and FTI/lovastatin co-treatment synergistically inhibit MM cell proliferation, even in the presence of IL-6 and IGF-1. Incomplete response to FTI treatment may be explained by alternative prenylation of K- and N-RAS by GGTase I in the presence of FTIs. As the majority of RAS mutations in MM occur in K- and N-RAS, FTI-resistance due to alternative geranylgeranylation may have therapeutic consequences.

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Analysis of PKB/Akt-Signaling and Oncogenic Ras in Multiple Myeloma.

Blood ◽

10.1182/blood.v114.22.831.831 ◽

2009 ◽

Vol 114 (22) ◽

pp. 831-831

Author(s):

Torsten Steinbrunn ◽

Thorsten Stühmer ◽

Manik Chatterjee ◽

Stefan Gattenlöhner ◽

Ralf C Bargou

Keyword(s):

Multiple Myeloma ◽

Cell Line ◽

Cell Lines ◽

Annexin V ◽

Akt Pathway ◽

Akt Inhibitor ◽

Mutation Status ◽

Ras Mutations ◽

Constitutive Activation ◽

Oncogenic Ras

Abstract Abstract 831 Introduction: Dysregulated signaling pathways contribute to cell growth, survival and drug resistance in multiple myeloma (MM). In about 50 percent of primary MM samples the protein kinase B (PKB)/Akt pathway appears to be constitutively active and its blockade strongly impairs survival, thus defining Akt-dependent versus Akt-independent MM. Because illegitimate activation of survival pathways can be a consequence of oncogenic Ras mutations, we analyzed the role of Ras in Akt-dependent primary MM samples. Methods: Primary MM samples from different patients (n=35) were tested for cell death induction with the small molecule Akt-inhibitor Akti 1/2 and the sensitivity was correlated to the mutation status of N- or K-Ras in these samples, which was obtained by RT-PCR. Immunohistochemistry was used to demonstrate the presence of phosphorylated Akt in corresponding patient biopsies. Apoptosis was measured by flow cytometry with Annexin V-APC or Annexin V-FITC and propidium-iodide staining. We further analyzed the consequences of Ras depletion with regard to Akt activation in a Ras wildtype (AMO-1) versus a K-Ras mutated (MM.1s) and an N-Ras mutated (INA-6) myeloma cell line using siRNA knockdown and Western blotting. Isoform specificity of the siRNAs was confirmed through the effects on ectopically expressed HA-tagged Ras proteins. Results: Of the 35 primary MM samples tested 17 (48.6%) showed sensitivity to pharmacologic inhibition of Akt and thus constituted the Akt-dependent myeloma subgroup. Activating mutations of either K-Ras (n=7) or N-Ras (n=7) were found in 14 samples (40%). However, we did not observe any obvious correlation between the mutation status of Ras and sensitivity to the Akt inhibitor, suggesting that oncogenic Ras is not a prerequisite for the constitutive activation of Akt. We extended our analysis to MM cell lines and performed isoform-specific siRNA mediated Ras knockdown. Depletion of K-Ras induced apoptosis selectively in K-Ras mutated MM.1s cells, while N-Ras mutated INA-6 cells were most sensitive to N-Ras knockdown. Contrarily, knockdown of either of these isoforms had no effect on survival in the Ras wildtype cell line AMO-1. Ras knockdown did not lead to a notable decrease in Akt(Ser473) phosphorylation, again indicating that oncogenic Ras signaling may not be mediated through the PKB/Akt-pathway in MM. Conclusion: Whereas MM cell survival might well be promoted by activating Ras mutations, oncogenic Ras does not appear to be instrumental in the constitutive activation of the PKB/Akt pathway in primary MM. Targeting Ras isoforms themselves can, however, lead to apoptosis in Ras mutated MM cell lines. Thus, even though oncogenic mutated Ras is currently not a druggable target, these experiments underscore that it is nonetheless desirable to develop ways to specifically block its function in MM. Disclosures: No relevant conflicts of interest to declare.

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