The role of RAS effectors in BCR/ABL induced chronic myelogenous leukemia

Abstract

BCR/ABL is the causative agent of chronic myelogenous leukemia (CML). Through structure/function analysis, several protein motifs have been determined to be important for the development of leukemogenesis. Tyrosine177 of BCR is a Grb2 binding site required for BCR/ABL-induced CML in mice. In the current study, we use a mouse bone marrow transduction/transplantation system to demonstrate that addition of oncogenic NRAS (NRASG12D) to a vector containing a BCR/ABL^Y177F mutant “rescues” the CML phenotype rapidly and efficiently. To further narrow down the pathways downstream of RAS that are responsible for this rescue effect, we utilize well-characterized RAS effector loop mutants and determine that the RAL pathway is important for rapid induction of CML. Inhibition of this pathway by a dominant negative RAL is capable of delaying disease progression. Results from the present study support the notion of RAL inhibition as a potential therapy for BCR/ABL-induced CML.

Keywords

BCR/ABL / chronic myelogenous leukemia (CML) / RAS / RAL

Introduction

The Philadelphia chromosome (Ph⁺), which is the result of a t(9;22) chromosomal translocation [1], is a hallmark of chronic myelogenous leukemia (CML) that results in the formation of the BCR/ABL oncogene [2]. Our research group and other researchers have determined the importance of several BCR/ABL protein motifs in the induction of CML, which include but are not limited to the N-terminal coiled coil (CC) domain [3,4], the ABL kinase domain [5,6], and tyrosine 177 (Y177) of BCR [7-10]. In a mouse bone marrow transduction and transplantation (BMT) model, BCR/ABL containing a tyrosine to phenylalanine mutation at Y177 (BCR/ABL^Y177F) is unable to induce myeloid leukemia [7,11]. Y177 is known to interact with the SH2 (src homology-2) domain of Grb2, which is an upstream activator of RAS.

RAS proteins belong to a family of small GTPases that mediate signals from cellular receptors. They switch from an inactive GDP-bound state to an active GTP-bound state by interacting with guanine nucleotide exchange factors (GEFs) such as Sos [12]. BCR/ABL recruits the adaptor protein Grb2 via Y177 [11]. Grb2 then recruits Sos to BCR/ABL, leading to the exchange of RAS-bound GDP for GTP. GTP-bound RAS is then able to activate its downstream effectors through its effector loop domain [13,14].

The effector loop region of RAS resides between amino acids 32 and 40 of the protein and directly interacts with downstream effectors that are involved in cell proliferation, apoptosis, and altered adhesion and migration. RAS effector loop mutants have been used to study the functions of particular pathways downstream of RAS in various cell types. The best studied effector pathways of RAS are RAF, PI3K, and RALGDS, which can be selectively activated by the RAS mutants T35S (RAF), E37G (RALGDS), and Y40C (PI3K), respectively [14,15].

The RAF pathway is involved in regulation of various cellular processes, including cell cycle regulation, proliferation, differentiation, and survival [16]. Activation of the MAPK cascade by RAF leads to translocation of ERK to the nucleus, wherein transcription factors can be regulated to effect changes in gene expression. Interestingly, mutations in BRAF have been found in about two thirds of melanomas and in subsets of thryoid and colon cancers [17]. The PI3K pathway has critical functions in cell proliferation, survival, metabolism, and protein translation [18,19].

RALGDS, a GEF for RAS-related protein (RAL), was one of the first RAS effectors to be identified [20]. However, the combination of its initially defined functions in vesicle trafficking and cell morphology and its lack of ability to transform NIH3T3 cells relegated it to the back burner [21] as studies on RAF and PI3K pathways and their potential functions in oncogenesis seemed considerably more attractive. Eventually, when studied in the right cellular context, the functions of RAL in transformation of NIH3T3 fibroblasts were observed [22,23]. Recent comparative analysis of tissue types has shown that two members of the RAL family, namely, RALA and RALB, are upregulated in tumors when compared with normal tissue samples [24-26].

The present study aims to examine the role of the three RAS effector pathways in the background of BCR/ABL^Y177F to determine which pathways, if any, are important for inducing CML-like myoproliferative disorder (MPD) in mice. We found that when any of the RAS effector mutants is expressed in the background of BCR/ABL^Y177F, mice developed CML-like MPD, albeit with different latencies. In particular, the RALGDS-activating mutant showed the most efficient rescue of CML-like MPD in the BCR/ABL^Y177F background, and inhibition of this pathway by a dominant negative RAL delayed disease progression. The present study demonstrates the importance of the RAL pathway in BCR/ABL-mediated leukemogenesis.

Materials and methods

DNA constructs

Construction of GFP, BCR/ABL, BCR/ABL^Y177F, and NRASG12D has been previously described [4,27,28]. BCR/ABL^Y177F-NRASG12D was generated by excising GFP from NRASG12D via 5′ and 3′ flanking EcoRI sites. The GFP was then replaced by ligation of an EcoRI-flanked BCR/ABL^Y177F.

The effector loop mutants were generated by PCR using a mega-primer strategy. A 5′ primer and a primer flanking the base pair to be changed (point mutation) were used to generate an approximately 120 bp long mega-primer. The second set of PCR was conducted using the mega-primer and the corresponding 3′ primer to amplify full-length NRASG12D with T35S, E37G, or Y40C substitutions. The specific base substitutions were ACC (threonine) to AGC (serine) for T35S, GAG (glutamic acid) to GGG (glycine) for E37G, and TAC (tyrosine) to TGC (cysteine) for Y40C. All effector domain mutants of NRAS were subsequently cloned into the murine stem cell virus (MSCV) retroviral vector under the control of the IRES element.

The BCR/ABL^Y177F-NRASG12D effector loop mutants were assembled in the same manner as BCR/ABL^Y177F-NRASG12D. pBabe-RALAS31N, pBabe-RALBS28N, and pBabe-RALGDS-CAAX were generously provided by Channing Der. We employed a PCR strategy to amplify the RAL constructs from the pBabe vector; these constructs were then inserted into the NotI-ClaI site downstream of BCR/ABL-GFP or BCR/ABL^Y177F-GFP and IRES but in-frame with a 2xmyc tag sequence. Previously published reports used a RALAS28N dominant negative mutant instead of an S31N mutant [29,30]; thus, a PCR primer was constructed to delete the three extra amino acids found in the N terminus of the RALAS31N mutant. NotI and ClaI restriction sites are underlined.

Primer sequences

RALAS28N 5′ primer- 5′AGC GGC CGC CAT GGC TGC AAA TAA GCC CAA

3′ primer- 5′CGC CAT CGA TTT ATA AAA TGC AGC ATC TTT CTTGA

RALBS28N 5′ primer- 5′AAG CGG CCG CCA TGG CTG CCA ACA A

3′ primer- 5′CGC CCA TCG ATT CAT AGT AAG CAA CAT CTT TCT TT

RALGDS-CAAX 5′ primer- 5′AAG CGG CCG CCA TGA TGG TAG ATT GCC G

3′ primer- 5′CGC CAT CGA TTC ACA TAA TTA CAC ACT TTG T

Cell culture and preparation of retrovirus

NIH3T3 and BOSC23 cells were maintained as previously described [28]. 32Dcl3 cells were grown in Dulbecco’s Modified Eagle’s Medium with 10% calf serum, 100 U/ml penicillin (GIBCO BRL, Grand Island, NY, USA), 100 µg/ml streptomycin (GIBCO BRL), and 10% WEHI-conditioned medium.

BOSC23 cells were used to produce retroviruses from the MSCV vectors described in “DNA Constructs.” Briefly, 1.5×10⁶ BOSC23 cells were transiently transfected with 5 μg of MSCV DNA via the calcium phosphate method [31]. Transfection media were replaced with fresh media about 12 h later. Retroviral supernatants were collected 24 h after the initial transfection.

Viral titers were determined by infecting 2×10⁵ NIH3T3 cells with varying amounts of retroviral supernatant as previously described [32]. Transduction units were calculated by multiplying the percentage of GFP⁺ cells in the dish by the number of cells present at the time of infection.

32D cells were infected by spinoculation. Briefly, 10⁶ 32Dcl3 cells were plated in 1 ml of complete media per well in a 6-well dish. To each well, 1 ml of retroviral supernatant and 8 μg/ml polybrene were added. The entire plate was centrifuged at 1200 rcf for 90 min at 25 °C. After centrifugation, cells were incubated at 37 °C for an additional 90 min. Cells were then washed with PBS and resuspended in media as described above. The GFP⁺ 32Dcl3 cells were sorted on a BD FACSAria sorter to greater than 98% purity.

Murine bone marrow transduction and transplantation model

Bone marrow cells were isolated, transduced, and transplanted as previously described [28,33,34]. Mice were analyzed for WBC counts, % GFP⁺ cells in the peripheral blood, and morphology of blood smears beginning at day 14 post-transplantation. For counting WBC, peripheral blood was diluted to 1:1000, treated with Zap-O-Globin (Coulter Diagnostics, Hialeah, FL, USA), and analyzed using a Coulter counter (Beckman Coulter, Fullerton, CA, USA). Survival curve analysis was carried out using StatView 5 (Abacus Concepts Inc., Berkeley, CA, USA).

Western blotting

32Dcl3 cells were starved of WEHI (0%) and FBS (0.1%) for 12 h prior to lysis. Cells were lysed at a concentration of 2.5×10⁷ cells/ml in lysis buffer [0.5% Igepal CA-630, 150 mM NaCl, 50 mM Tris-HCl pH 8.0, 1 mM EDTA, 1× Complete Protease Inhibitor (Roche), 1 μg/ml pepstatin A, 1 μg/ml leupeptin, 1 mM Na₃VO₄, 1 mM PMSF, 10 mM NaF] for 15 min on ice. Post-nuclear supernatants were collected after centrifugation at 14 000 rpm for 15 min at 4 °C. Lysates were stored at -70 °C. Afterward, 4× SDS loading buffer (200 mM Tris-HCl pHβ6.8, 10% 2-ME, 40% glycerol, 8% SDS, 0.4% bromophenol blue) was added to thawed lysates at 1× concentration and lysates were boiled for 5 min before loading on a 6%-18% gradient gel. Proteins were transferred to nitrocellulose membranes via wet transfer.

Membranes were probed with the following primary antibodies: anti-Abl (AB3) from the conditioned medium of a hybridoma cell line, anti-Dynamin (BD Biosciences, San Jose, CA, USA), anti-myc tag (9b11) (Cell Signaling Technologies, Beverly, MA), and anti-phosphotyrosine clone 4G10 (Upstate Biotechnology, Lake Placid, NY, USA). Secondary HRP-conjugated goat-anti-mouse or goat-anti-rabbit antibodies obtained from Pierce Biotechnology (Rockford, IL, USA) were used to visualize proteins using Piere Super Signal West Femto chemiluminescence reagents.

Flow cytometry

Peripheral blood or tissue cell suspensions were treated with red blood cell lysis buffer [ACK: 150 mM NH₄Cl, 1 mM KHCO₃, 0.1 mM Na₂EDTA (pH 7.3)], washed in FACS buffer (PBS, 2% FBS), and resuspended in fresh FACS buffer. Cells were blocked with anti-mouse CD16/CD32 (F_c block, 2.4G2, BD Pharmingen, San Jose, CA, USA) and stained with the following BD Pharmingen antibodies: APC-Mac1 (M1/70), PE-Gr1 (RB6-8C5), APC-B220 (RA3-6B2), PE-CD19 (1D3), APC-Thy1.2 (30-H12), PE-CD3ϵ, and APC-c-Kit (2B8). Flow cytometry was performed on a FACSCalibur, and data were analyzed using FlowJo (Tree Star, Ashland, OR, USA).

Immunofluorescence staining and microscopy

NIH3T3 cells grown on fibronectin-treated coverslips were fixed in 3% paraformaldehyde/PBS. Cells were permeabilized in 0.5% Igepal (NP-40)/PBS, washed in 0.1% Igepal/PBS, and stained with anti-Abl (Ab3) diluted to 1:50 in 0.1% Igepal/PBS for 1 h. To visualize Abl proteins, cells stained with an AlexaFluor594 conjugated Goat-anti-Mouse secondary antibody (Invitrogen, Carlsbad, CA, USA) were diluted to 1:250 in 0.1% Igepal/PBS. Coverslips were mounted on slides using Vectashield mounting media with DAPI (Vector Laboratories, Burlingame, CA, USA). Cells were visualized using a Leica TCS SP2 Spectral Confocal Microscope ATOF attached to a Leica DMIRC-2 inverted microscope at a resolution of 63×under oil immersion.

Results

Expression of oncogenic NRAS in the presence of BCR/ABL^Y177F results in CML-like MPD

Considering that Y177 of BCR/ABL is a Grb2 binding site and Grb2 is an upstream activator of RAS, we investigated whether or not oncogenic NRASG12D could “rescue” the CML phenotype in the presence of BCR/ABL^Y177F. We designed a bicistronic retroviral vector containing a BCR/ABL^Y177FGFP fusion gene and a myc-tagged oncogenic N-RAS (BCR/ABL^Y177F-NRASG12D) (Fig. 1A). The resulting vector, along with previously described controls [9,11,27,28], was then used in a murine bone marrow transduction/transplantation (BMT) experiment. The expression of BCR/ABL and myc-tagged NRASG12D proteins was confirmed by infection of 32D cells and subsequent Western blotting (data not shown).

All animals, except for the GFP controls, died within 4 months. Mice receiving wild-type BCR/ABL succumbed to CML-like MPD between days 20 and 24 (Fig. 1B). The mice showed high white blood cell (WBC) counts (>100 000 cells/mm³), pulmonary hemorrhage, and hepatosplenomegaly because of infiltration of Mac-1⁺/Gr-1⁺ granulocytic cells (data not shown). FACS analysis of the bone marrow (BM) also showed an expansion of Mac-1⁺/Gr-1⁺ cells (Fig. 1C). Mice receiving BCR/ABL^Y177F died between days 68 and 120 (Fig. 1B) and exhibited moderately increased WBC counts (<30 000 cells/mm³) with no significant enlargement of the spleen or liver and normal lungs. All of the mice developed thymic or lymph node tumors containing Thy1.2⁺/CD3e⁺ cells expressing GFP but showed no expansion of granulocytic cells (Fig. 1C). In addition, very few circulating GFP⁺ cells were found, which may be due to tumor site homing.

Mice receiving NRASG12D alone developed either acute myeloid leukemia (AML) or chronic myelomonocytic leukemia (CMML)-like MPD between days 35 and 90 (Figs. 1B and 1C), as previously shown [27]. Three out of 10 mice exhibited a CMML-like disease characterized by hepatosplenomegaly, increased WBC numbers (<100 000 cells/mm³), and expanded granulocytic and monocytic cell lineages (data not shown). The remaining seven mice exhibited an AML-like disease characterized by a slight increase in WBC number (50 000 cells/mm³), moderate hepatosplenomegaly, and an expansion of immature granulocytic cells (Fig. 1C).

In contrast to the results seen in mice receiving BCR/ABL^Y177F or NRASG12D alone, the CML-like MPD phenotype manifested by wild-type BCR/ABL alone was observed in mice receiving BCR/ABLY177F-NRASG12D, albeit with a longer latency. The mice died between days 35 and 70 (Fig. 1C) because of pulmonary hemorrhage, exhibited gross hepatosplenomegaly and showed WBC counts>150 000 cells/mm³ (data not shown). FACS analysis showed an infiltration of Mac-1⁺/Gr-1⁺ myeloid cells into the spleen and liver (data not shown) and an expansion of this population in the bone marrow (Fig. 1C). Therefore, activation of RAS and its subsequent binding to downstream effectors is an important and necessary step in the pathogenesis of BCR/ABL-induced CML.

Activation of single RAS effector pathways is insufficient to induce myeloid leukemia

To determine the functions of different downstream effectors of RAS on leukemogenesis, we utilized three well-characterized RAS effector loop mutants [14,18,35]. We constructed effector-loop mutants in the NRASG12D background and cloned them into the MSCV retroviral vector. Bone marrow cells isolated from 5-fluorouracil-treated mice were then infected with retroviruses containing NRASG12D/35S, NRASG12D/37G, and NRASG12D/40C, along with NRASG12D (positive control) or vector alone (negative control), and subsequently transplanted into lethally irradiated recipient mice. The mice were monitored weekly from day 14 after transplantation onward by scanning for GFP⁺ cells. During the initial scans, the percentage of GFP⁺ cells in the peripheral blood was found to be higher in mice expressing NRASG12D/37G (30%-40%) than the GFP percentage in the NRASG12D/T35S and NRASG12D/Y40C mice, which were similar to values of control mice (15%-20%) (Fig. 2A).

During subsequent evaluations, the percentage of GFP⁺ cells increased exponentially in the NRASG12D mice but remained more or less the same in the NRASG12D/37G, NRASG12D/35S, NRASG12D/40C, and vector control mice (data not shown). Consequently, all mice expressing NRASG12D succumbed to an AML- or CMML-like disease between days 32 and 50, while mice expressing the effector domain mutants, as well as the vector control, remained alive for the entire duration of observation (3 months) (Fig. 2B).

The above results suggest that none of the NRAS partial loss-of-function mutants is sufficient to induce myeloid leukemia in mice with the same efficiency as NRASG12D.

RAS effector loop mutants induce CML-like MPD in the BCR/ABL^Y177F background

To further elucidate relevant leukemia-related pathways downstream of NRAS, we sought to combine BCR/ABL^Y177F with the NRAS effector domain mutants and use these constructs in our mouse BMT model.

Constructs were designed in the same manner as the BCR/ABL^Y177F-NRASG12D depicted in Fig. 1A, replacing the NRASG12D with one of three NRAS effector domain double mutants: NRASG12D/35S, NRASG12D/37G, or NRASG12D/40C. Expression of both BCR/ABL and NRASG12D proteins was determined by Western blotting (Fig.β3A).

Consistent with the expected results, mice receiving BCR/ABL transduced bone marrow died of rapid CML-like MPD between days 16 and 18 (Fig. 3B). The mice exhibited high WBC counts (100 000 cells/mm³ to 200 000 cells/mm³), massive hepatosplenomegaly caused by infiltration of Mac-1⁺/Gr-1⁺ cells, and pulmonary hemorrhages (data not shown). As observed in the previous experiment and shown in Figs.β1B andβ1C, BCR/ABL^Y177F-NRASG12D mice also died of CML-like MPD (Fig. 2.3B and data not shown). Also, as previously observed, mice receiving BCR/ABL^Y177F remained healthy for the longest period and eventually succumbed to T cell leukemia/lymphoma (Fig. 3B, data not shown).

All mice receiving the different BCR/ABL^Y177F-NRASG12D effector mutants developed CML-like MPD but with different latencies (Figs. 3B and 3C). Surprisingly, NRASG12D/37G was found to be the most efficient in inducing CML in mice. The above results suggest that activation of the RALGDS pathway has an important function in the pathogenesis of CML.

Activation of RALGDS is important for efficient BCR/ABL-mediated leukemogenesis

The finding that BCR/ABL^Y177F-NRASG12D/37G induced CML-like MPD faster than any of the other NRAS effector domain mutants led us to further examine the functions of the RALGDS pathway in BCR/ABL-mediated leukemogenesis. Previous studies showed that an activated RALA (G23V or Q72L mutant) can transform HEK-HT cells, as assayed by soft-agar colony growth [25], and that siRNA depletion of RALB induces apoptosis in human tumor cell lines [26]. To test the effects of manipulation of the RAL pathway on the induction of BCR/ABL-mediated leukemogenesis, we designed retroviral vectors for use in our BMT assay. New DNA vectors containing wild-type BCR/ABL plus RALA or RALB containing a dominant negative (DN) S28N mutation were designed (Fig. 4A). RALS28N mutants preferentially bind GDP and remain in this inactive state [36]. To complement this study, we also designed retroviral vectors comprised of BCR/ABL^Y177F and a RALGDS appended with a C-terminal RAS-CAAX domain (BCR/ABL^Y177F-RALGDS-CAAX). RALGDS is a guanine nucleotide exchange factor for RAL that is activated by directly interacting with the RAS effector loop. Addition of the CAAX domain allows for constitutive localization of RALGDS at the plasma membrane, which is important for RAL activation [37]. All proteins were highly expressed in 32D cells, as assayed by Western blotting (data not shown).

BCR/ABL^Y177F-RALGDS-CAAX was introduced into our mouse BMT system along with relevant controls. All BCR/ABL mice died between days 17 and 20 of typical CML-like MPD. After 70 days, all the mice that received BCR/ABL^Y177F remained alive and healthy. However, three out of 10 mice that received BCR/ABL^Y177F-RALGDS-CAAX died of CML-like MPD and showed highly elevated WBC counts (>200 000 cells/mm³) and hepatosplenomegaly because of infiltration by granulocytes. An abundance of these granulocytic cells was also found in the bone marrow (data not shown). Except for one mice, all the remaining seven mice showed increasing numbers of WBCs (>50 000 cells/mm³) but remained alive at day 70.

To determine whether or not the presence of a DN RAL can delay induction of BCR/ABL-mediated leukemogenesis, we altered our mouse BMT protocol. Considering that the constructs inhibited a cellular pathway with an unknown function in hematopoietic cell homing, we determined a window of time in which we could infect 5-FU treated donor bone marrow, inject it into lethally irradiated recipient mice, and allow for proper homing to the bone marrow niche before the gene of interest was fully expressed. Previous studies have determined that initial homing to the hematopoietic microenvironment takes approximately 3 h and that long-term hematopoiesis is established at 20 h post-injection [38]. We determined that GFP takes approximately 8 h post-infection to express in NIH3T3 cells but that robust expression of GFP is not seen in 5-FU treated BM cells until over 24 h post-infection (Fig.βS1). To minimize the effect of the DN RAL on bone marrow homing, we altered our BMT protocol by decreasing the bone marrow infection time to 12 h and slightly increasing the viral titers. The viral titers of BCR/ABL and BCR/ABL-RALB/S28N were well matched (~2.5×10⁵ TU).

Mice that received BCR/ABL transduced bone marrow in our modified BMT assay died of CML-like MPD beginning at day 19, which was around the same time that mice undergoing the normal BMT protocol tended to become sick. BCR/ABL mice died of pulmonary hemorrhages and had slightly elevated WBC counts (>100 000 cells/mm³) and hepatosplenomegaly because of granulocyte infiltration (data not shown). The above results confirm that our modified BMT protocol results in the same disease phenotype that we previously observed. Addition of DN RALB caused a minor delay in disease onset of approximately 2 days (Fig. 4C). These mice also died of CML-like MPD identical to that seen in the BCR/ABL mice. Overall, the data presented above show that RAL has an important function in efficient induction of CML by BCR/ABL.

Discussion

In the present report, we examined the function of RAS effectors in mediating BCR/ABL-induced CML. Grb2 binding-deficient BCR/ABL (BCR/ABL^Y177F) is defective in activating the RAS pathway and is unable to induce CML-like MPD in a mouse model. Addition of activated RAS rescues this defect, and all mice receiving bone marrow transduced with BCR/ABL^Y177F-NRASG12D die rapidly of CML-like MPD. When NRASG12D is replaced with a double mutant that selectively activates a single downstream effector pathway, all mutants were found to be capable of inducing CML-like MPD. However, disease onset occurs faster upon activation of the RALGDS pathway. In addition, coexpression of dominant negative RALB was found to ameliorate BCR/ABL-induced CML in mice. These results are consistent with the finding that downregulation of RALA by miR-181a or RNAi leads to growth inhibition and apoptosis of CML cells [39,40]. Overall, the data demonstrate the importance of RAL family proteins in mediating BCR/ABL-induced leukemogenesis.

Expression of BCR/ABL in a mouse bone marrow transduction/transplantation model induces rapid and fatal CML-like MPD that resembles human chronic phase CML in 100% of the recipient mice [28,33]. Mutating Y177of BCR to phenylalanine completely removes this ability, and mice receiving BCR/ABL^Y177F-infected bone marrow instead succumb to T-lymphoid leukemia after a latency period ranging from 3 months to 4 months (Fig. 1 and Refs. [4,11]). Y177 is part of a Grb2 binding site in the N-terminal domain of BCR that leads to the activation of RAS [41].

In the same mouse model, activated NRASG12D efficiently induces either AML or CMML-like MPD [27]. However, when mutations affecting the effector loop affinity for RAF, PI3K, or RALGDS pathways are introduced, NRASG12D loses its ability to induce hematologic malignance (Fig. 2), which may be due to the requirement of cooperation of multiple RAS effector pathways to induce hematologic insult.

The combination of BCR/ABL^Y177F and NRASG12D leads to rapid induction of fatal CML-like MPD in lethally irradiated recipient mice. BCR/ABL has been determined to be a potent activator of RAS, and, unsurprisingly, addition of activated NRAS to a Grb2 binding deficient BCR/ABL rescues the CML phenotype that is observed in mice transplanted with wild-type BCR/ABL. Interestingly, out of the three RAS effector pathways examined herein, the RALGDS pathway resulted in considerably faster rescue of the CML phenotype than either the RAF or PI3K pathways.

The three known members of the RALGEF family include RALGDS, RGL, and RLF [42-44]. RALGDS is dispensable for embryonic development, as demonstrated by the generation of viable and fertile RALGDS knockout mice. The loss of RALGDS is possibly compensated for by the presence of the two remaining RAL GEFs. However, when these mice are used in a model of skin carcinogenesis, tumor size and load are decreased [45], which suggests that RALGDS could have an important function in survival signaling that is not redundant in RGL or RLF. The above results, taken together with the findings presented in the current study, suggest that RALGDS could be a potential target for therapeutic intervention in CML and possibly other cancers.

References

[1] Rowley JD. Letter: a new consistent chromosomal abnormality in chronic myelogenous leukaemia identified by quinacrine fluorescence and Giemsa staining. Nature1973; 243(5405): 290-293

[2] Groffen J, Stephenson JR, Heisterkamp N, de Klein A, Bartram CR, Grosveld G. Philadelphia chromosomal breakpoints are clustered within a limited region, bcr, on chromosome 22. Cell1984; 36(1): 93-99

[3] McWhirter JR, Galasso DL, Wang JY. A coiled-coil oligomerization domain of Bcr is essential for the transforming function of Bcr-Abl oncoproteins. Mol Cell Biol1993; 13(12): 7587-7595

[4] Zhang X, Subrahmanyam R, Wong R, Gross AW, Ren R. The NH(2)-terminal coiled-coil domain and tyrosine 177 play important roles in induction of a myeloproliferative disease in mice by Bcr-Abl. Mol Cell Biol2001; 21(3): 840-853

[5] Cortez D, Kadlec L, Pendergast AM. Structural and signaling requirements for BCR-ABL-mediated transformation and inhibition of apoptosis. Mol Cell Biol1995; 15(10): 5531-5541

[6] Lugo TG, Pendergast AM, Muller AJ, Witte ON. Tyrosine kinase activity and transformation potency of bcr-abl oncogene products. Science1990; 247(4946): 1079-1082

[7] He Y, Wertheim JA, Xu L, Miller JP, Karnell FG, Choi JK, Ren R, Pear WS. The coiled-coil domain and Tyr177 of bcr are required to induce a murine chronic myelogenous leukemia-like disease by bcr/abl. Blood2002; 99(8): 2957-2968

[8] Million RP, Harakawa N, Roumiantsev S, Varticovski L, Van Etten RA. A direct binding site for Grb2 contributes to transformation and leukemogenesis by the Tel-Abl (ETV6-Abl) tyrosine kinase. Mol Cell Biol2004; 24(11): 4685-4695

[9] Sattler M, Mohi MG, Pride YB, Quinnan LR, Malouf NA, Podar K, Gesbert F, Iwasaki H, Li S, Van Etten RA, Gu H, Griffin JD, Neel BG. Critical role for Gab2 in transformation by BCR/ABL. Cancer Cell2002; 1(5): 479-492

[10] Goga A, McLaughlin J, Afar DE, Saffran DC, Witte ON. Alternative signals to RAS for hematopoietic transformation by the BCR-ABL oncogene. Cell1995; 82(6): 981-988

[11] Million RP, Van Etten RA. The Grb2 binding site is required for the induction of chronic myeloid leukemia-like disease in mice by the Bcr/Abl tyrosine kinase. Blood2000; 96(2): 664-670

[12] Campbell SL, Khosravi-Far R, Rossman KL, Clark GJ, Der CJ. Increasing complexity of Ras signaling. Oncogene1998; 17(11 11 Reviews): 1395-1413

[13] Deininger MW, Goldman JM, Melo JV. The molecular biology of chronic myeloid leukemia. Blood2000; 96(10): 3343-3356

[14] White MA, Nicolette C, Minden A, Polverino A, Van Aelst L, Karin M, Wigler MH. Multiple Ras functions can contribute to mammalian cell transformation. Cell1995; 80(4): 533-541

[15] Campbell PM, Singh A, Williams FJ, Frantz K, Ulkü AS, Kelley GG, Der CJ. Genetic and pharmacologic dissection of Ras effector utilization in oncogenesis. Methods Enzymol2006; 407: 195-217

[16] Leicht DT, Balan V, Kaplun A, Singh-Gupta V, Kaplun L, Dobson M, Tzivion G. Raf kinases: function, regulation and role in human cancer. Biochim Biophys Acta2007; 1773(8): 1196-1212

[17] Roberts PJ, Der CJ. Targeting the Raf-MEK-ERK mitogen-activated protein kinase cascade for the treatment of cancer. Oncogene2007; 26(22): 3291-3310

[18] Rodriguez-Viciana P, Warne PH, Khwaja A, Marte BM, Pappin D, Das P, Waterfield MD, Ridley A, Downward J. Role of phosphoinositide 3-OH kinase in cell transformation and control of the actin cytoskeleton by Ras. Cell1997; 89(3): 457-467

[19] Downward J. Targeting RAS signalling pathways in cancer therapy. Nat Rev Cancer2003; 3(1): 11-22

[20] Albright CF, Giddings BW, Liu J, Vito M, Weinberg RA. Characterization of a guanine nucleotide dissociation stimulator for a ras-related GTPase. EMBO J1993; 12(1): 339-347

[21] Feig LA. Ral-GTPases: approaching their 15 minutes of fame. Trends Cell Biol2003; 13(8): 419-425

[22] White MA, Vale T, Camonis JH, Schaefer E, Wigler MH. A role for the Ral guanine nucleotide dissociation stimulator in mediating Ras-induced transformation. J Biol Chem1996; 271(28): 16439-16442

[23] Urano T, Emkey R, Feig LA. Ral-GTPases mediate a distinct downstream signaling pathway from Ras that facilitates cellular transformation. EMBO J1996; 15(4): 810-816

[24] Lim KH, O’Hayer K, Adam SJ, Kendall SD, Campbell PM, Der CJ, Counter CM. Divergent roles for RalA and RalB in malignant growth of human pancreatic carcinoma cells. Curr Biol2006; 16(24): 2385-2394

[25] Lim KH, Baines AT, Fiordalisi JJ, Shipitsin M, Feig LA, Cox AD, Der CJ, Counter CM. Activation of RalA is critical for Ras-induced tumorigenesis of human cells. Cancer Cell2005; 7(6): 533-545

[26] Chien Y, White MA. RAL GTPases are linchpin modulators of human tumour-cell proliferation and survival. EMBO Rep2003; 4(8): 800-806

[27] Parikh C, Subrahmanyam R, Ren R. Oncogenic NRAS rapidly and efficiently induces CMML- and AML-like diseases in mice. Blood2006; 108(7): 2349-2357

[28] Zhang X, Ren R. Bcr-Abl efficiently induces a myeloproliferative disease and production of excess interleukin-3 and granulocyte-macrophage colony-stimulating factor in mice: a novel model for chronic myelogenous leukemia. Blood1998; 92(10): 3829-3840

[29] Tchevkina E, Agapova L, Dyakova N, Martinjuk A, Komelkov A, Tatosyan A. The small G-protein RalA stimulates metastasis of transformed cells. Oncogene2005; 24(3): 329-335

[30] Omidvar N, Pearn L, Burnett AK, Darley RL. Ral is both necessary and sufficient for the inhibition of myeloid differentiation mediated by Ras. Mol Cell Biol2006; 26(10): 3966-3975

[31] Pear WS, Nolan GP, Scott ML, Baltimore D. Production of high-titer helper-free retroviruses by transient transfection. Proc Natl Acad Sci USA1993; 90(18): 8392-8396

[32] Gross AW, Zhang X, Ren R. Bcr-Abl with an SH3 deletion retains the ability to induce a myeloproliferative disease in mice, yet c-Abl activated by an SH3 deletion induces only lymphoid malignancy. Mol Cell Biol1999; 19(10): 6918-6928

[33] Pear WS, Miller JP, Xu L, Pui JC, Soffer B, Quackenbush RC, Pendergast AM, Bronson R, Aster JC, Scott ML, Baltimore D. Efficient and rapid induction of a chronic myelogenous leukemia-like myeloproliferative disease in mice receiving P210 bcr/abl-transduced bone marrow. Blood1998; 92(10): 3780-3792

[34] Daley GQ, Van Etten RA, Baltimore D. Induction of chronic myelogenous leukemia in mice by the P210bcr/abl gene of the Philadelphia chromosome. Science1990; 247(4944): 824-830

[35] Joneson T, White MA, Wigler MH, Bar-Sagi D. Stimulation of membrane ruffling and MAP kinase activation by distinct effectors of RAS. Science1996; 271(5250): 810-812

[36] Hinoi T, Kishida S, Koyama S, Ikeda M, Matsuura Y, Kikuchi A. Post-translational modifications of Ras and Ral are important for the action of Ral GDP dissociation stimulator. J Biol Chem1996; 271(33): 19710-19716

[37] Matsubara K, Kishida S, Matsuura Y, Kitayama H, Noda M, Kikuchi A. Plasma membrane recruitment of RalGDS is critical for Ras-dependent Ral activation. Oncogene1999; 18(6): 1303-1312

[38] Plett PA, Frankovitz SM, Orschell CM. Distribution of marrow repopulating cells between bone marrow and spleen early after transplantation. Blood2003; 102(6): 2285-2291

[39] Fei J, Li Y, Zhu X, Luo X. miR-181a post-transcriptionally downregulates oncogenic RalA and contributes to growth inhibition and apoptosis in chronic myelogenous leukemia (CML). PLoS ONE2012; 7(3): e32834

[40] Zhu X, Li Y, Luo X, Fei J. Inhibition of small GTPase RalA regulates growth and arsenic-induced apoptosis in chronic myeloid leukemia (CML) cells. Cell Signal2012; 24(6): 1134-1140

[41] Chu S, Li L, Singh H, Bhatia R. BCR-tyrosine 177 plays an essential role in Ras and Akt activation and in human hematopoietic progenitor transformation in chronic myelogenous leukemia. Cancer Res2007; 67(14): 7045-7053

[42] de Bruyn KM, de Rooij J, Wolthuis RM, Rehmann H, Wesenbeek J, Cool RH, Wittinghofer AH, Bos JL. RalGEF2, a pleckstrin homology domain containing guanine nucleotide exchange factor for Ral. J Biol Chem2000; 275(38): 29761-29766

[43] Shao H, Andres DA. A novel RalGEF-like protein, RGL3, as a candidate effector for rit and Ras. J Biol Chem2000; 275(35): 26914-26924

[44] Wolthuis RM, Bos JL. Ras caught in another affair: the exchange factors for Ral. Curr Opin Genet Dev1999; 9(1): 112-117

[45] González-García A, Pritchard CA, Paterson HF, Mavria G, Stamp G, Marshall CJ. RalGDS is required for tumor formation in a model of skin carcinogenesis. Cancer Cell2005; 7(3): 219-226

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