RIT1 mutations in myeloid neoplasia
Gómez-Seguí et al. 2013
Supplementary Appendix
NOVEL RECURRENT MUTATIONS IN THE RAS-LIKE GTP-BINDING GENE RIT1 IN MYELOID
MALIGNANCIES
Inés Gómez-Seguí MD, Hideki Makishima MD, PhD, Andrés Jerez MD, PhD, Kenichi Yoshida MD,
BartlomiejPrzychodzen BS, Satoru Miyano, Yuichi Shiraishi, Holleh D. Husseinzadeh, MD, Kathryn
Guinta BS, Michael Clemente BS, Naoko Hosono MD, PhD, Michael A. McDevitt MD, PhD, Alison R.
Moliterno, MD, PhD, MikkaelA. Sekeres, MD, MS,
Seishi Ogawa,MD. PhD, and Jaroslaw P.
MaciejewskiMD, PhD.
A.Supplementary Methods
B. Supplementary Information
C. Supplementary Figures and Figure Legends
D. Supplementary Tables
E. Supplementary Literature Cited
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A. Supplementary Methods
Patient population
Bone marrow aspirates or peripheral blood samples were collected from 722 patients seen at Cleveland
Clinic and Johns Hopkins University School of Medicine (Supp.Table 1). Informed consent for sample
collection was obtained according to protocols approved by the individual Institutional Review Boards and
in accordance with the Declaration of Helsinki. Diagnosis was confirmed and assigned according to World
Health Organization (WHO) classification criteria.1 Lower-risk MDS was defined as patients having <5%
myeloblasts. Patients with >5% myeloblasts constituted those with higher-risk disease. To study the
germline genotype, immunoselected CD3+ lymphocytes were used. Cytogenetic analysis was performed
according to standard banding techniques based on 20 metaphases.Clinical parameters studied included
age, gender, hemoglobin, leukocytes, neutrophils, monocytes, platelets, bone marrow blasts, cytogenetics,
IPSS risk group2, MDAPS risk group3 or CALGB cytogenetic risk group4 and RIT1 mutation/amplification
status. Follow-up of patients was updated on July 2012 and all follow-up data were censored at that point.
The median follow-up of surviving patients was 21.3 months (range, 1 to 144 months).
Cytogenetics and single nucleotide polymorphism (SNP) array analyses
Technical details regarding sample processing for SNP array assays were previously described.5,6
Affymetrix 250K and SNP 6.0 arrays(Affymetrix, Santa Clara, CA) were used. A stringent algorithm was
applied for the identification of SNP-A lesions. Patients with SNP array lesions concordant with metaphase
cytogenetics or typical lesions known to be recurrent required no further analysis. Changes reported in our
internal or publicly-available (Database of Genomic Variants; http://projects.tcag.ca/variation) copy number
variation databases were considered non-somatic and excluded. Results were analyzed using CNAG
(v3.0)7 or Genotyping Console (Affymetrix). All other lesions were confirmed as somatic or germline by
analysis of CD3-sorted cells.8
Whole exome sequencing
Whole exome sequencing was performed as previously reported.9 Briefly, tumor DNAs were extracted from
patients’ bone marrow or peripheral blood mononuclear cells. For germline controls, DNA was obtained
from either paired CD3 positive T cells with or without prior culture in the presence of phytohemagglutinin
and IL-2. Whole exome capture was accomplished based on liquid phase hybridization of sonicated
genomic DNA having 150 - 200bp of mean length to the bait cRNA library synthesized on magnetic beads
(SureSelect®, Agilent Technology), according to the manufacture’s protocol. SureSelect Human All Exon
50Mb kit was used for 20 cases. The captured targets were subjected to massive sequencing using
IlluminaGAIIx and/or HiSeq 2000 with the pair end 75-108 bp read option, according to the manufacture’s
instruction. The raw sequence data generated from IlluminaGAIIx or HiSeq2000 sequencers were
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Gómez-Seguí et al. 2013
processed through the in-house pipeline constructed for whole-exome analysis of paired cancer genomes
at the Human Genome Center, Institute of Medical Science, University of Tokyo, which are summarized in
a previous report.9 The data processing is divided into two steps,
1) Generation of a .bam file (http://samtools.sourceforge.net/) for paired normal and tumor samples for
each case.
2) Detection of somatic point mutations and indels by comparing normal and tumor BAM files.
Alignment of sequencing reads on hg19 was visualized using Integrative Genomics Viewer (IGV) software
(http://www.broadinstitute.org/igv/).10
Sanger sequencing and allele-specific PCR analysis
Exons of selected genes were amplified and underwent direct genomic sequencing by standard techniques
on the ABI 3730xl DNA analyzer (Applied Biosystems, Foster City, CA) as previously described.11-13 The
allelic presence of T244A, T244G and T245G was also determined by allele-specific PCR.Primers for RIT1
sequencing and RIT1 allele-specific PCR are provided in Supp.Table6 and PCR conditions will be provided
if requested. Mutations were detected by bidirectional sequencing and scored as pathogenic if not present
in non-clonal paired CD3-derived DNA.RIT1 mutations were named after the NCBI transcript reference
sequence NM_006912.5. Coding and sequenced exons to explore concomitant mutations in patients with
RIT1 mutations/amplifications are shown in Supp.Table 7.
Quantitative RT-PCR by TaqMan probes
Total RNA was extractedfrom bone marrow mononuclear cells and cell lines. cDNA was synthesized from
500 ng total RNA using the SuperScript® III First-Strand Synthesis System (Invitrogen). Quantitative gene
expression levels were detected using real-time PCR with the ABI PRISM 7500 Fast Sequence Detection
System and FAM dye labeled TaqMan MGB probes (Applied Biosystems). TaqMan assays were performed
according to the manufacturer’s instructions. The expression level of target genes was normalized to
GAPDH mRNA. Primers and probes for all genes analyzed were purchased from Applied Biosystems gene
expression assays products (RIT1: Hs00608424_m1; BAD: Hs00188930_m1; BCL2: Hs00608023_m1;
MYC: Hs00153408_m1; and GAPDH: Hs99999905_m1).
Statistical analysis of clinical data
Chi-square and Fisher’s exact tests were used to analyze differences in the distribution of categorical
variables among patient subsets. Mann–Whitney–Wilcoxon non-parametric U-test was used to analyze
differences in mean ranks. Unadjusted time-to-event analyses were performed using the Kaplan–Meier
estimate and for comparisons, log-rank tests. For multivariate analyses, a Cox proportional hazards model
was conducted. Variables considered for model inclusion were age, gender, hemoglobin, leukocytes,
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Gómez-Seguí et al. 2013
neutrophils, monocytes, platelets, bone marrow blasts, cytogenetics, IPSS risk group, MDAPS risk group or
CALGB risk group and RIT1 mutation/amplification status. All computations were performed using the
statistical package SPSS, version 17.0 (SPSS Inc., Chicago, IL, USA). A two-sided P value below .05 was
considered significant.
Publicly available databases and analytical tools
The February 2009 human reference sequence (GRCh37) produced by the Genome Reference
Consortium was used as reference genome (UCSC genome browser; http://genome.ucsc.edu/cgibin/hgGateway).Expression array data was extracted from Oncomine (https://www.oncomine.org/). Somatic
mutation data was searched by Catalogue of somatic mutations in cancer (COSMIC) database in Welcome
Trust Sanger Institution website (http://www.sanger.ac.uk/genetics/CGP/cosmic/) and The Cancer Genome
Atlas (TCGA) (http://cancergenome.nih.gov/). Each potential mutation was compared against databases of
known SNPs, including Entrez Gene (http://www.ncbi.nlm.nih.gov/gene) and the Ensemble Genome
Browser (http://useast.ensembl.org/index.html).
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B. Supplementary Information
Somatic RIT1 mutations and amplifications in myeloid malignancies
We estimated the allelic frequency of the two pilot cases analyzed by NGS to be 35-40%, consistent with
70-80% of the clone size present in heterozygous mutations. Subsequently, we sequenced all 5 coding
exons of theRIT1 gene (exons 2-6) in a cohort of 221 patients; 3additional mutant cases were found. All
mutations were located in the same aminoacid (F82) in exon 5, other exons were unaffected. These
findings prompted us to further expand the screening of mutations in exon 5 to a total of 722 patients with
myeloid malignancies, including 200 primary AML cases available for open access data through
TCGA(Supplementary Table 1).All together, a total of 9 mutations in 9 patients were found (Supplementary
Table 2): in 6 cases the aminoacid F82 was affected (3 F82C, and 1 of each F82I, F82L and F82V), the
E81 residue in two cases (E81G and E81Q) and an M90I mutation was found in one patient (TCGA data
base). All mutations were located in the Switch II domain of this protein, an effector region very close to the
GTP-binding site ‘G3’ that transposes upon activation by GTP and is conserved among species (Fig. 1). All
human leukemic cell lines tested showed WT RIT1 (Supp.Table 8).
RIT1 belongs to the family of Ras GTPases, which are best known for their ability to serve as molecular
switches regulating cell growth, differentiation and survival, mainly through the MEK/ERK and PI3K/AKT
pathways.14 Gene mutations that result in expression of constitutively active forms of Ras have been linked
to oncogenesis in animal models and humans.15 RIT1 shares high sequence identity (>50%) with the Ras
subfamily of GTPases, although it has a highly conserved but distinct G2 effector region and lacks the
CAAX motif necessary for plasma membrane association;16 however, Rit is plasma membrane-localized
due to a C-terminal cluster of basic amino acids.16 Due to these minor differences from the main Ras family
members, Rit can signal both common and unique Ras-responsive elements, even in a cell type-dependent
manner, e.g. Rit fails to activate p38-, JNK- or ERK-MAPK or PI3K/Akt pathways in fibroblasts (NIH3T3
cells),17 but is able to stimulate proliferation, differentiation and survival through most of these pathways in
neurons (PC6 cells).18-20 Rit1 has also been implicated in promoting stress-dependent cell survival18 and to
be critical in the survival of neurons after brain injury.21 Consistent with these results, Rit knockout mice
displayed increased apoptosis and selective disruption of MAPK signaling in response to oxidative
stress.21,22 and Rit blockade in PC6 shRNAi-treated cells significantly increased apoptosis18 and expression
of dominant-negative Rit inhibited neuronal-growth factor-induced neurite outgrowth.19
An experimental form of Rit1 protein carrying the Q79L mutation has been reported to result in neuronal
differentiation morphologically different to that caused by oncogeneic Ras23, and inhibits the apoptosis
subsequent to growth factor withdrawal in PC6 cells.19 Fibroblasts expressing Rit79L display strong growth
transformation and rapid proliferation and even induce tumors in nude mice at sites of injection, showing
the tumorigenic phenotype of this activating mutation.17 This mutation, similar (but not identical) to the
human somatic mutations found by us in myeloid neoplasms, is also located in the switch II domain
adjacent to the GTP-binding site G3, and appears analogous to the Q61 mutation in other Ras family
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proteins such as NRAS or KRAS, but none of the leukemogeneic mutations affected the corresponding
residue (Q79) in our cohort. RIT1 has been reported to be both mutated and amplified in hepatocarcinoma.
Interestingly, amplification of RIT1 was found in a quarter of HCC cases (11/43) and an additional case
(5%) carried the E81G mutation, identical to one of the cases reported in this study.24,25 Other mutations in
breast basal carcinoma (n=1), melanoma (n=1) and rectal adenocarcinoma (n=1) can be found in the
COSMIC database (Welcome Trust Sanger Institute), although these,unlike the mutations reported in our
manuscript, were found in amino acids located toward the N-terminus of the protein (P199P, R112C and
D216Y).
The canonical nature of RIT1 mutations, coinciding with amplification of the RIT1 locus through whole or
partial chromosome duplication in patients with similar phenotype, along with overexpression of putative
downstream effectors in patients with Rit abnormalities and its reported transforming capability in in vitro
models, suggest that these are activating mutations.In fact, Ras proteins are one of the most common gainof-function mutations found in human cancers26 and novel transforming Ras mutations have been
described recently in myeloid cancers outside the known 12,13 and 61 codons, that exhibited oncogenic
properties in comparison with wild-type Ras in biochemical and functional assays.27
By the number of affected reads, the RIT1 clone appeares to be dominant; serial testing showed the
presence of the RIT1 mutation at initial diagnosis (Suppl. Fig. 1), suggesting that RIT1 may be of ancestral
origin. This is supported by the fact that the Nras mutation is sufficient to induce a CMML-like disease in
mouse models.28 Parallel analyses using SNP-arrays and sequencing demonstrated that mutations were
heterozygous with the exception of one case, which showed a 1q amplification involving the RIT1 locus: in
this case the mutant allele was duplicated (Supp. Fig. 2). In 2 cases, the 1q amplification was serially
studied: while the post-PV myelofibrosis case showed 1q+ as an acquired event associated with fibrotic
transformation, the case of primary myelofibrois displayed it from an early stage (Suppl. Fig. 1), in
agreement with a relatively frequent observation of trisomy 1 in primary MF29 and post-PV MF.30 While Ras
mutations are rare events in MF and may predict leukemic transformation,31 we did not find RIT1 mutations
in classical MPN cases tested. Of note is that the common amplified region of 1q in our cohort was big and
involved several candidate genes.
RIT1 expression and downtream events
The RIT1 gene, like most Ras-related proteins, is ubiquitously expressed,16 including within hematologic
tissues, as is shown in publicly available expression array data on lymphocytes (NK, B and T), myeloid
cells, monocytes, whole blood and CD34+ progenitors, together with some cell lines of leukemia and
lymphoma (MOLT4, K562, DAUDY, HL60) (GeneNote and BioGPS databases). We directly analyzed the
expression of the RIT1 gene in cases harboring mutations; cDNA sequencing revealed that mutant allele
was expressed equally or even at higher levels than the WT (Suppl Fig. 2). qRT-PCR showed no difference
in the expression levels of RIT1 mRNA between mutant and wild-type (WT) cases (median relative ratio of
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Gómez-Seguí et al. 2013
0.58 in mutant vs. 0.18 in WT cases; P=0.098), but cases with 1q+ showed significant overexpression
(median relative ratio 1.29 vs. 0.18, P=0.041; Fig. 2A). We also explored RIT1 expression in publicly
available expression arrays performed in the spectrum of myeloid malignancies (Haferlach leukemia
dataset of Oncomine database). The average expression of RIT1 mRNA was increased in AML with
complex karyotype and chronic myeloid leukemia (CML) (relative expression 2.2 and 2.3, respectively, vs.
1.8 in healthy donors; P=.016 and P<.001, respectively) (Supp. Table 5). Based on expression of 74
controls, upmodulation of RIT1 (>mean+2 SD of control expression) was found in 9/824 cases (1.1%),
remarkably, in 1/38 AML with MLL abnormalities, 3/351 AML with normal karyotype or karyotype with less
than two abnormalities, 2/206 MDS and 3/76 CML.
RIT1 is a central regulator of a p38 MAPK-dependent signaling cascade that functions as a critical cellular
survival mechanism in response to various stress stimuli.18 In particular, RIT1 is able to activate
phosphorylation of AKT inhibiting apoptosis, and to activate the classical MAPK cascade regulating
proliferation.17,18,22 We therefore studied the expression of putative RIT1 downstream effector genes
(i.e.BAD and BCL2, involved in regulation of apoptosis through the AKT pathway, and MYC regulation of
proliferation through the classical MAPK pathway) in 6 patients carrying RIT1 mutations and/or
amplifications, 7 patients with WT RIT1 as well as healthy donors. Four out of 6 patients (67%) with RIT1
abnormalities showed a lower expression of the BAD gene when compared to healthy donors (median
relative BAD expression (median relative expression 0.54vs. 0.98 in healthy donors; P=0.064; although
patients with RIT1 WT also had downregulation of the BAD gene (median relative expression 0.43 vs. 0.98
in healthy donors; P= 0.046). Similarly, 3/6 (50%) patients with RIT1 abnormalities had a higher expression
of BCL2 (median relative expression 2.88 vs.1.04 for healthy donors; P=0.086). Similarly, patients with
RIT1 WT also tended to display upregulation of the BCL2 gene (median relative expression 2.96 vs. 1.04 in
healthy donors; P= 0.143). These data were suggestive of a more pronounced AKT pathway activation in
most of the patients with RIT1 abnormalities (Suppl.Fig.3B). When MYC gene expression was investigated,
patients carrying RIT1 abnormalities showed elevated MYC mRNA levels (median relative MYC expression
2.28 vs. 0.93, in mutants vs. healthy donors, respectively; P=.053), but not patients with WT RIT1 (median
relative expression 1.42; P=0.248), suggestive of a stronger proliferative drive in cases with abnormal RIT1
(Suppl.Fig.3B).
Molecular and clinical associations of RIT1 mutations and amplifications
To clarify the molecular association of RIT1 mutations with other genetic defects, we screened mutations in
any of the Ras genes in an expanded cohort of patients studied with myeloid malignancies (n=333) (Supp
Table 9 and Supp Table 10). We then expanded the study of concomitant mutations by traditional methods
to 7 patients with RIT1 mutations and 4 with 1q amplification (n=10, 1 patient with both mutation and
amplification) with available samples for screening of mutations of 17 well-known genes in myeloid cancers
(i.e. TET2, IDH1, IDH2, DNMT3A, CBL, RUNX1, ASXL1, UTX (KDM6A), EZH2, NRAS, KRAS, TP53,
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JAK2, SF3B1, U2AF1, SRSF2, and SETBP1). In one atypical RIT1 mutant case with 1q amplification in
SNP array we also found a concomitant KRAS mutation, but such a combination was not found in any other
patient (Supp. Fig. 1B).
The distribution of patients carrying RIT1 mutations or RIT1 amplifications within various malignancies is
shown in Fig. 2A. Interestingly, no mutation or amplification was found in lower-grade forms of MDS.
Although the diversity of diagnosis and the relatively low frequency of RIT1 abnormalities made it difficult to
find discrete clinical correlations, we did discover that RIT1 abnormalities were significantly more frequent
in CMML patients than in other patients (56% vs. 9%, respectively; P=0.001) and also were associated with
abnormalities in chromosome 7 (33% vs. 6%, respectively; P= 0.017) (Supp. Table 3).
When cases with WT RIT1 and those with mutations were compared, mutant and amplified cases showed
a trend towards having a shorter median overall survival (19 vs. 14 months, P=0.053). This difference
achieved statistical significance when selecting patients with MDS or MDS/MPN diagnosis (24 vs. 16
months, P=0.029; Fig. 2C and Supp. Table 4). However, in multivariate analyses, RIT1 abnormalities lost
its association with poor prognosis, which was correlated with older age, higher leukocyte count,
hemoglobin level, adverse cytogenetics, or CMML MD Anderson Prognostic Score (MDAPS) (Supp. Table
4A). RIT1 abnormalities did not predict for AML progression. (Supp. Table 4B).
Ras mutations are believed to confer a poor prognosis in other solid tumors, but the real impact on
outcome is still a matter of controversy in leukemia and may differ between disease subtype; for example,
in AML it confers either a poor prognosis, or has no clear prognostic significance in the largest series,32
whereas in JMML it may represent a good-risk marker.33 In our series, patients carrying RIT1 anomalies
appeared to have a poor risk, but this may be due to the occurrence of these anomalies in the high-risk
subgroups (e.g. patients with chromosome 7 abnormalities).
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C. Supplementary Figures and Figure Legends
Supplementary Figure 1(A). Ancestrality of RIT1 mutations and amplifications. Four cases with
available sample at different evolutive stages are shown. In UPN 1 and UPN 6 the mutation can be seen at
diagnosis, during follow-up and also at the time of AML evolution, all of them apparently in heterozygosis.
In UPN15, the log2 ratio of the copy number probes shows gain of 1q at an early stage of myelofibrosis and
after years of evolution. In UPN18, 1q amplification cannot be seen in the Polycythemia Vera (PV) stage
and appears clearly in the myelofibrotic (MF) phase. (B) Circos diagram showing mutations associated with
RIT1 mutants (red) or RIT1 amplifications (orange). One patient carried both a mutation in RIT1 and KRAS
and beside, the SNP-A showed RIT1 amplification.
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Supplementary Figure 2(A). Concomitance of RIT1 amplification and mutation in one patient (UPN4). (B).
Mutated allele is equally expressed relative tothe referenced allele, analyzed by parallel genomic DNA and
cDNA sequencing.
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Gómez-Seguí et al. 2013
Supplementary Figure 3(A).Relative RIT1 expression in the spectrum of myeloid neoplasms represented
in box plots (data analyzed from Oncomine database). Blue bars represent the percentage of each
subgroup expressing RIT1 mRNA levels over the median+2SD of the healthy donors. (B) Relative
expression of BAD, BCL2 and MYC, all putative downstream effectors of RIT1, in patients with RIT1
activation (by mutations or locus amplifications) and healthy donors.
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Gómez-Seguí et al. 2013
Supp. Table 1. Cohort of patients screened for RIT1 mutations and amplifications.
Diagnosis WHO 2008
Myeloproliferative Neoplasms
Chronic Myeloid Leukemia
RIT1
RIT1
Sequence; N mutations; amplifications;
(SNP-A; N)
N (%)
N (%)
163 (129)
0 (0)
4 (3)
33 (2)
0 (0)
0 (0)
Chronic Phase
13 (1)
0 (0)
0 (0)
Accelerated Phase
5 (1)
0 (0)
0 (0)
Blast Crisis
15 (0)
0 (0)
0 (0)
Polycythemia Vera (PV)
34 (34)
0 (0)
0 (0)
Post-PV Myelofibrosis
12 (11)
0 (0)
2 (18)
Essential Thrombocytemia (ET)
42 (42)
0 (0)
0 (0)
Post-ET Myelofibrosis
12 (11)
0 (0)
1 (9)
Primary Myelofibrosis
30 (27)
0 (0)
1 (4)
131 (105)
5 (4)
2 (2)
Chronic Myelomonocytic Leukemia
72 (50)
5 (7)
2 (4)
Juvenile Myelomonocytic Leukemia
37 (36)
0 (0)
0 (0)
Refractory Anemia with Ring Sideroblasts with Thrombocytosis
10 (8)
0 (0)
0 (0)
AtypicalChronicMyeloidLeukemia
3 (3)
0 (0)
0 (0)
Myelodisplastic/MyeloproliferativeNeoplasm, Unclassifiable
9 (8)
0 (0)
0 (0)
119 (79)
1 (1)
2 (3)
5 (4)
0 (0)
0 (0)
13 (11)
0 (0)
0 (0)
2 (1)
0 (0)
0 (0)
Refractory Anemia with Ring Sideroblasts
13 (13)
0 (0)
0 (0)
Refractory Cytopenia with Mmultilineage Dysplasia
38 (18)
0 (0)
0 (0)
Refractory Cytopenia with Excess of Blasts - type 1
22 (13)
0 (0)
1 (8)
Refractory Cytopenia with Excess of Blasts - type 2
26 (19)
1 (4)
1 (5)
64 (44)
1 (2)
1 (2)
245 (201)
2 (1)
0 (0)
with t(8;21) or inv(16)
24 (18)
0 (0)
0 (0)
with t(15;17)
21 (20)
0 (0)
0 (0)
with normal karyotype
117 (97)
1 (1)
0 (0)
with other abnormalities
55 (41)
0 (0)
0 (0)
with complex karyotype
28 (25)
1 (4)
0 (0)
722 (558)
9 (1)
9 (2)
Myelodisplastic/MyeloproliferativeNeoplasms
Myelodisplastic Syndromes
Myelodisplastic Syndrome with isolated del(5q)
Refractory Anemia
Refractory Cytopenia with Unilineage Dysplasia
Secondary Acute Myeloid Leukemia
Primary Acute Myeloid Leukemia
Total
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Supp. Table 2. Main characteristics of patients carrying RIT1 mutations and amplifications.
UPN
Age
Gender
Diagnosis
Karyotype
Nucleotide
Change
Aminoacid
change
SNP-A gain
Chr. Position
Size (Mb)
Prognosis
score
1
67
M
CMML-2
46,XY.
c.T244A
p.F82I
-
-
-
2
79
F
CMML1
45,XY,-7.
c.T244G
p.F82V
-
-
3
81
M
CMML-1
45XY,del(5q),-7.
c.T246A
p.F82L
-
-
4
68
M
CMML-1
46,XY,+1,der(1;15)(q10;q10).
c.A242G
p.E81G
1q21.1 - q44
5
56
M
CMML-2
46,XY.
c.T245G
p.F82C
-
OS
Status
MDAPS: Int-1
26mo
DEAD
-
MDAPS: Int-2
6mo
DEAD
-
MDAPS: Int-2
5mo
DEAD
144083908 - 247185974
103.1
MDAPS: Int-2
AML 16moafter
diagnosis
39mo
DEAD
-
-
MDAPS:
High
Refractorydisease
4mo
ALIVE
AML 35moafter
diagnosis
37mo
DEAD
2mo
DEAD
14mo
DEAD
6
70
M
RAEB-2
46,XY.
c.G241C
p.E81Q
-
-
-
IPSS:Int-2
7
85
F
AML-MRC
46,XX,t(1;3)(p36;q21).
c.T245G
p.F82C
-
-
-
IPSS: High
8
43
F
AML- M5B
9
47
F
Disease
progression
Relapse
10moafter
diagnosis
46,XX.
c.T245G
p.F82C
-
-
-
CALGB: Int
AML- M2
Complex (+8, 7q-, 5q-)
C.G270A
p.M90I
-
-
-
CALGB: Poor
12mo
DEAD
-
-
1p11.1 - q44
121137730 - 247170176
126.03
IPSS: High
6mo
DEAD
1mo
DEAD
36mo
DEAD
32mo
DEAD
16mo
DEAD
10
63
M
AML-MRC
46,XY,+1,der(1;7)(q10;p10),
t(5;22)(q13;q11.2).
11
82
F
RAEB2
49-53,XX,+X,+1,del(5q),+10,+11,+18,+21,+22.
-
-
trisomy 1
-
-
IPSS: High
12
59
F
CMML-1
46,XX,der(16)t(1;16)(q12;q11.2).
-
-
1q11.2 - qter
120863833 - 245326460
124.46
MDAPS: Int-2
AML 19 moafter
diagnosis
13
65
F
CMML-1
47,XX,+9.
-
-
1q21.1 - q44
143734641 - 247190999
103.46
MDAPS: Int-2
14
58
F
RAEB-1
46,XX,del(5q),+9,add(16)(q24).
-
-
1q21.1 - q44
145231749 - 246948674
101.72
IPSS:Int-2
15
58
M
PMF
46,XY,-7,+9,+16,-21,t(1;?).
-
-
1q21.1 - q44
142756696 - 247110269
104.35
-
AML 16 y. after
diagnosis
16 y.
DEAD
16
51
F
Post-PV MF
not done
-
-
1q12 - q44
142215052 - 244658857
102.44
-
MF 35 yafterPV
diagnosis
4 y.
ALIVE
-
MF 20 y. after ET
diagnosis
6 y.
ALIVE
-
MF 22 y. after PV
diagnosis
3 y.
DEAD
17
18
60
67
M
M
Post-ET MF
Post-PV MF
not done
not done
-
-
1q21.1 - q32.1
1q21.2 - q44
142559711 - 202429502
147409484 - 245326460
59.87
97.92
Chr.:chromosome; Mb: megabase; OS: overall survival; mo: months; CMML: chronic myelomonocytic leukemia; RAEB: Refractory Anemia with Excess of Blasts; AML-MRC: Acute Myeloid Leukemia with Myelodisplasia Related Changes; PMF:
primary myelofibrosis; MF: myelofibrosis; PV: Policytemia Vera; ET: Essential Trombocytemia; MDAPS: MD Anderson Prognosis Score for CMML; IPSS: International Prognostic Scoring System for MDS; CALGB: The Cancer and Leukemia
Group B.
13
RIT1 mutations in myeloid neoplasia
Gómez-Seguí et al. 2013
Supp. Table 3. Clinical characteristics of myeloid malignancies with or without RIT1 mutations.
Age at diagnosis
Range
Gender (male)
Leukocytes (median, x106/L)
Hemoglobin (median, g/dL)
Platelets (median, x106/L)
BoneMarrowBlasts (%)
WHO-2008 diagnosis
ChronicMyelomonocyticLeukemia
Refractory Cytopenia with Excess of Blasts type 2
AML with myelodisplasia related changes
primary AML
Karyotype
normal karyotype
complexkaryotype
monosomy 7/7qRisk category
High risk IPSS (for MDS cohort)(Int-2/high)
High risk MDAPS (for CMML cohort)(Int-2/high)
High risk CALGB classification (for AML)
RIT1 WT
n (%)
RIT1
mutant (%)
713 (98.8)
61.3 ± 15
18 - 88
207 (40.4)
9 (1.2)
66.2 ± 15
43 - 85
4 (44.4)
5.11
9.6
11.6
9.3
0.360
0.909
76
11
48
9
0.349
0.714
67 (9.4)
25 (3.5)
61 (8.5)
245 (34.3)
5 (55.6)
0.001
1 (11.1)
0.273
1 (11.1)
0.556
2 (22.2)
0.356
241 (48.1)
67 (13.4)
31 (6.2)
4 (44.4)
0.549
1 (11.1)
0.658
0.017
174 (52.9)
40 (60.6)
48 (20.1)
2 (100)
3 (33.3)
5 (100)
1(50)
P value
0.342
0.530
0.284
0.094
0.366
14
RIT1 mutations in myeloid neoplasia
Gómez-Seguí et al. 2013
Supp. Table 4. Survival analyses of patients with RIT1 WT vs. RIT1 with abnormalities
(amplifications or mutations).
A) Overall Survival Analyses
Median OverallSurvival (months)
MDS/MDS-MPN/AML patients
MDS/MDS-MPN patients
High grade MDS (RAEB, AML-MRC)
RAEB
RAEB-2
AML-MRC
CMML
primary AML
Cox Regression Analysis for Overall Survival
High grade MDS (n=94)
Cytogenetic Risk (IPSS2)
Hemoglobin<10g/dL
RAEB (n= 47)
Hemoglobin<10g/dL
CytogeneticRisk (IPSS2)
RAEB2 (n= 24)
CytogeneticRisk (IPSS2)
AML-MRC (n= 51)
CytogeneticRisk (IPSS2)
6
Leukocytecount>20 x10 /L
CMML (n= 66)
MD Anderson Prognostic Score3
Cytogenetic Risk (Such et al.34)
Gender (female)
primary AML
Age>60 years
CytogeneticRisk (CALGB4)
RIT1 WT
RIT1 mut/amp
Pvalue
n= 490
19
24
12
15
13
10
17
16
HR
n= 14
14
16
6
16
0.5
2.4
26
12
CI 95%
0.053
0.029
0.351
0.612
0.603
0.184
0.594
0.473
P value
1.82
1.06
(1.35  2.45)
(1.17  3.63)
<0.001
0.012
3.43
1.70
(1.37  8.57)
(1.07  2.69)
0.009
0.025
2.48
(1.20  5.12)
0.014
2.08
3.43
(1.37  3.16)
(1.47  7.98)
0.001
0.004
1.79
1.58
0.46
(1.25  2.56)
(1.02  2.44)
(0.22  0.95)
0.003
0.021
0.037
2.82
1.52
(1.92  4.14)
(1.14  2.03)
<0.001
0.004
15
RIT1 mutations in myeloid neoplasia
Gómez-Seguí et al. 2013
B) Time to AML progression analyses
AML progression (%)
RAEB
CMML
Time to 25% of AML progression (months)
RAEB
CMML
Cox Regression Analysis for AML Progression
RAEB (n=47)
Hemoglobin<10 g/dL
CMML (n=66)
MD Anderson Prognostic Score
RIT1 WT
RIT1 mut/amp
P value
n= 490
n=14
31.8
14.5
33.3
28.6
0.694
0.309
19
29
35
16
0.961
0.511
HR
CI 95%
P value
4.12
(1.10 - 15.44)
0.036
2.11
(1.07 - 4.12)
0.030
Supp.Table 5.Comparison of median relative mRNA expression of RIT1 in different myeloid
malignancies assessed by expression array (Oncomine database).
Diagnosis
Median
Relative RIT1
expression
P value*
healthydonors (n=74)
1,8494
CBF-AML (n=68)
2,0215
0.732
APL (n=37)
AML with NK or other abnormalities
(n=351)
1,6323
0.215
1,8331
0.279
AML with MLL abnormalities (n=38)
1,9446
AML with CK (n=48)
2,2081
0.815
0.016
MDS (n=206)
1,8305
0.937
CML (n=76)
* compared to healthy donors.
2,3284
<0.001
16
RIT1 mutations in myeloid neoplasia
Gómez-Seguí et al. 2013
Supp. Table 6. Primers for RIT1 sequencing of coding exons, cDNA of exon 5 and allelespecific PCR for mutant alleles in aminoacid F82.
RIT1 Primers for coding exons
5' - 3' sequence
Exon2-3_Forward
GAGGGACAGGCCAGAATATG
Exon2-3_Reverse
ACCAACTGCTGATACCCTTG
Exon4-5_Forward
TTTTAAATAGGCATTTCTTCCG
Exon4-5_Reverse
CCAAGAATCTGTAAGCCAAGAAAC
Exon6_Forward
ACTCCAATCTGGGCAACAAG
Exon6_Reverse
TGAACTTAGTCAAACACACATGG
RIT1 Primers for coding exons
cDNA_Forward
ATCAGCCACCGATTCCCAGAAGAT
cDNA_Reverse
ATCGTCAGTACGTCGGACTCGATA
RIT1 Allele specific PCR (T244A)
RIT1_T244A_FI
ATGACCCTTGTTTCCCTCTAGGCAGTGA
RIT1_T244A_RI
TCATATACTGGTCCCGCATGGCTGTTAA
RIT1_T244A_FO
TGGACATTTTGGATACAGCTGGACAGGT
RIT1_T244A_RO
TGACTTGTTTCCCACAAGAACCACAGGT
RIT1 Allele specific PCR (T244G)
RIT1_T244G_FI
ATGACCCTTGTTTCCCTCTAGGCAGGGT
RIT1_T244G_RI
CATATACTGGTCCCGCATGGCTGTGAC
RIT1_T244G_FO
GGCTTTTTTACTCTAGCAAAGGGGAGGGG
RIT1_T244G_RO
TCCCACAAGAACCACAGGTGTATCGTCA
RIT1 Allele specific PCR (T245G)
RIT1_T245G_FI
TGACCCTTGTTTCCCTCTAGGCAGAATG
RIT1_T245G_RI
CATATACTGGTCCCGCATGGCTGCAA
RIT1_T245G_FO
GGCTTTTTTACTCTAGCAAAGGGGAGGGG
RIT1_T245G_RO
TCCCACAAGAACCACAGGTGTATCGTCA
17
RIT1 mutations in myeloid neoplasia
Gómez-Seguí et al. 2013
Supp. Table 7. List of genes and their annotation screened by Sanger sequencing or
ARMS-PCR
Amino
Coding Sequenced
Genes
EnsemblecDNAsequence
acids
exons
exons
TET2
ENST00000380013
2002
3-11
3-11
IDH1
ENST00000345146
414
3-10
4
IDH2
ENST00000330062
452
2-11
4
DNMT3A
ENST00000264709
912
2-23
21-23
CBL
ENST00000264033
906
1-16
8, 9
RUNX1
ENST00000300305
480
2-9
2-9
ASXL1
ENST00000375687
1541
1-13
13
KDM6A (UTX)
ENST00000377967
1401
1-29
1-29
EZH2
ENST00000320356
751
2-20
2-20
NRAS
ENST00000369535
189
2-5
2
KRAS
ENST00000311936
188
2-5
2
JAK2
ENST00000381652
1132
2-24
13*
TP53
ENST00000269305
393
2-11
5-8
SETBP1
ENST00000282030
1596
2-6
2-6
SF3B1
ENST00000335508
1304
1-25
14-15
U2AF1
ENST00000291552
240
1-8
2, 6
221
1-2
1
SRSF2
ENST00000392485
* JAK2 V617F was screened by ARMS-PCR.
Supp. Table 8. Mutational status of RIT1 gene in 17 human leukaemia cell lines
Cell line
CMK-2
GDM-1
HEL
HL60
KASUMI
KG-1
KMS-12
KU-812
MEG-01
MEG-A2
MOLM-13
MUTZ-8
NKM-1
SKM-1
TF-1
THP-1
UT-7
RIT1exon 4-5
mutational status
WT
WT
WT
WT
WT
WT
WT
WT
WT
WT
WT
WT
WT
WT
WT
WT
WT
18
RIT1 mutations in myeloid neoplasia
Gómez-Seguí et al. 2013
Supp. Table 9. Diagnosis distribution of the cohort studied by whole exome sequencing.
Diagnosis
Total
AML (n=200)
lowrisk
38
Intermediate
127
high-risk
35
MDS (n=65)
low grade
44
25
high grade
MDS/MPN (n=36)
24
CMML
12
other MDS/MPN
secondary AML (n=32)
TOTAL
333
Therapy-related condition (n=19)
19
RIT1 mutations in myeloid neoplasia
Gómez-Seguí et al. 2013
Supp. Table 10. Mutations found in patients studied by whole exome sequencing and carrying Ras
family mutations showed in Figure 2D.
Gene
Patient 1 (sAML)
NRAS
BCOR
DNMT3A
IDH1
STAG2
Patient 2 (MDS)
NRAS
NRAS
ASXL1
BCOR
DNMT3A
RUNX1
Patient 3 (AML)
NRAS
DNMT3A
FLT3
IDH1
NPM1
Patient 4 (AML)
NRAS
DNMT3A
NPM1
Patient 5 (AML)
NRAS
DNMT3A
ETV6
IDH2
Patient 6 (AML)
NRAS
DNMT3A
NPM1
CEBPA
PTPN11
WT1
Patient 7 (AML)
NRAS
DNMT3A
EZH2
IDH1
Patient 8 (AML)
NRAS
DNMT3A
NPM1
NM number
Genome Position
Mutation
NM_002524
NM_001123383
NM_153759
NM_005896
NM_001042751
115258747
39934196
25462009
209113113
123220476
p.G12D
p.S135fs
p.G611S
p.R132S
p.R1045X
NM_002524
NM_002524
NM_015338
NM_017745
NM_175629
NM_001001890
115258744
115258748
31023070
39922019
25467449
36171602
G13D
G12S
S852X
V1351fs
G543C
L294fs
NM_002524
NM_022552
NM_004119
NM_005896
NM_002520
115258744
25457243
28592642
209113112
170837547
p.G13D
p.R882C
p.D835Y
p.R132H
p.W288fs
NM_002524
NM_022552
NM_002520
115258744
25468163
170837547
p.G13D
p.E505*
p.W288fs
NM_002524
NM_022552
NM_001987
NM_002168
115258744
25463163
12043874
90631934
p.G13V
splice_site
splice_site
p.R140Q
NM_002524
NM_022552
NM_002520
NM_004364
NM_002834
NM_024426
115258744
25468133
170837547
33792294
112939981
32417907
p.G13D
p.Q515*
p.W288fs
p.R343fs
p.I545L
p.A382fs
NM_002524
NM_022552
NM_004456
NM_005896
115258747
25463529
148506443
209113113
p.G12D
p.P718L
p.R690H
p.R132C
NM_002524
NM_022552
NM_002520
115258748
25463286
170837547
p.G12C
p.R736H
p.W288fs
20
RIT1 mutations in myeloid neoplasia
Gene
NM number
Patient 9 (AML)
NRAS
NM_002524
DNMT3A
NM_022552
DNMT3A
NM_022552
U2AF1
NM_001025203
Patient 10 (AML)
NRAS
NM_002524
TET2
NM_017628
TET2
NM_017628
U2AF1
NM_001025203
Patient 11 (AML)
NRAS
NM_002524
RUNX1
NM_001754
STAG2
NM_001042749
NM_017628
TET2
NM_017628
TET2
Patient 12 (AML)
NRAS
NM_002524
NPM1
NM_002520
IDH1
NM_005896
Patient 13 (AML)
NRAS
NM_002524
Patient 14 (AML)
NRAS
NM_002524
Patient 15 (MDS)
NRAS
NM_002524
PDGFRA
NM_006206
Patient 16 (JMML)
NRAS
NM_002524
Patient 17 (sAML)
NRAS
NM_002524
ZRSR2
NM_005089
Patient 18 (AML)
NRAS
NM_002524
CSF3R
NM_156039
Patient 19 (AML)
NRAS
NM_002524
CUL1
NM_003592
Patient 20 (MDS)
NRAS
NM_002524
Patient 21 (AML)
NRAS
NM_002524
Patient 22 (AML)
NRAS
NM_002524
Patient 23 (AML)
NRAS
NM_002524
CEBPA
NM_004364
Patient 24 (AML)
NRAS
NM_002524
TP53
NM_000546
Gómez-Seguí et al. 2013
Genome Position
Mutation
115256528
25463271
25469029
44524456
p.Q61H
p.A741V
p.E477*
p.S34F
115256529
106157969
106156747
44524456
p.Q61R
p.Q958fs
p.R550*
p.S34F
115256529
36231783
123220476
106196213
106183007
p.Q61P
p.R201*
p.R1045*
p.R318*
splice-site
115258747
170837547
209113113
p.G12D
p.W288fs
p.R132C
115258748
p.G12C
115256529
p.Q61L
115258747
55129915
p.G12D
p.C150F
115258748
p.G12C
115258748
15836719
p.G12S
p.N261Y
115258744
36933434
p.G13D
p.T618I
115256528
148454181
p.Q61H
p.N141S
115258744
p.G13D
115256530
p.Q61K
115256530
p.Q61K
115258747
33792395
p.G12D
p.309in_frame_insV
115258747
7577081
p.G12D
p.E286G
21
RIT1 mutations in myeloid neoplasia
Gene
NM number
Patient 25 (AML)
NRAS
NM_002524
NPM1
NM_002520
Patient 26 (AML)
KRAS
NM_033360
DNMT3A
NM_022552
NPM1
NM_002520
U2AF1
NM_001025203
Patient 27 (AML)
KRAS
NM_033360
DNMT3A
NM_022552
IDH2
NM_002168
Patient 28 (AML)
KRAS
NM_033360
DNMT3A
NM_022552
NPM1
NM_002520
PDGFRA
NM_006206
Patient 29 (AML)
KRAS
NM_033360
DNMT3A
NM_022552
IDH2
NM_002168
ASXL1
NM_015338
Patient 30 (CMML)
KRAS
NM_033360
ATM
NM_000051
SRSF2
NM_003016
TET2
NM_017628
Patient 31 (CMML)
KRAS
NM_033360
CSF3R
NM_000760
NF1
NM_000267
TET2
NM_001127208
TET2
NM_001127208
Patient 32 (CMML)
KRAS
NM_033360
ASXL1
NM_015338
CBL
NM_005188
EZH2
NM_004456
LUC7L2
NM_016019
U2AF35
NM_006758
Patient 33 (sAML)
KRAS
NM_033360
Patient 34 (AML)
KRAS
NM_033360
SRSF2
NM_003016
Patient 35 (AML)
KRAS
NM_033360
Patient 36 (AML)
KRAS
NM_033360
Gómez-Seguí et al. 2013
Genome
Position
Mutation
115258744
170837547
p.G13D
p.W288fs
25398284
25467032
170837547
44524456
p.G12V
p.Q615*
p.W288fs
p.S34Y
25378562
25467106
90631934
p.A146T
p.G590fs
p.R140Q
25398281
25457242
170837547
55156567
p.G13D
p.R882H
p.W288fs
p.G990R
25380282
25457242
90631838
31023271
p.A59E
p.R882H
p.R172K
p.S921fs
25378562
108155188
74732959
106157684
p.A146T
p.L1327fs
p.P95L
p.L862fs
25398262
36932224
29684326
106164897
106156747
p.L19F
p.Q749X
p.R?*
p.Y1255X
p.R550X
25398306
31022592
119149003
148506205
139060903
44514777
p.K5E
p.R693X
p.W408L
p.K718fs
splice-site
p.Q157R
25398284
p.G12D
25398284
74732389
p.G12V
p.173in_frame_insKS
25398281
p.G13D
25398284
p.G12V
22
RIT1 mutations in myeloid neoplasia
Gene
NM number
Patient 37 (AML)
KRAS
NM_033360
SETBP1
NM_015559
Patient 38 (AML)
KRAS
NM_033360
SF3B1
NM_012433
Patient 39 (CMML)
RIT1
NM_006912
RUNX1
NM_001122607
SF3B1
NM_012433
STAG2
NM_001042751
STAG2
NM_001042751
TET2
NM_017628
Patient 40 (CMML)
RIT1
NM_006912
Patient 41 (MDS)
PRPF8
NM_006445
RIT1
NM_006912
STAG2
NM_006603
Patient 42 (AML)
EGFR
NM_005228
RIT1
NM_006912
TP53
NM_000546
TP53
NM_000546
Gómez-Seguí et al. 2013
Genome
Position
Mutation
25398211
42531907
p.I36M
p.D814N
25398284
198266834
p.G12D
p.K700E
155874287
36252940
198267491
123197737
123197737
106158302
p.F82I
p.S114X
p.E622D
p.E621fs
p.E621K
p.Q1068fs
155874285
p.F82L
1565301
155874286
123224540
p.M1307I
p.F82C
p.R1131fs
55238900
155874261
7578414
7578206
p.T638M
p.M90I
p.V173fs
p.S215G
23
RIT1 mutations in myeloid neoplasia
Gómez-Seguí et al. 2013
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