Research paperExamination of clinically-derived p210 BCR/ABL1 RhoGEF mutations in a murine bone marrow transplantation model of CML
Introduction
The Philadelphia chromosome (Ph) is the product of a reciprocal translocation that fuses 5′ sequences of the BCR gene on chromosome 22, with 3′ sequences of the ABL1 gene on chromosome 9. Depending on where the breakpoint falls in chromosome 22, a variable amount of BCR is fused with ABL1. This results in the production of several different sizes of BCR-ABL1 fusion proteins which are associated with distinct clinical sequelae [1]. Thus, p210 BCR/ABL1 is observed in 96 % of CML and one-third of Ph + acute lymphoblastic leukemia (ALL), while p190 BCR/ABL1 is present in two-thirds of Ph + ALL. If left untreated patients with Ph + CML will progress to a blast crisis (BC) that shares clinical features with Ph + ALL. All BCR/ABL1 variants contain a constitutive tyrosine kinase activity within the ABL1 sequences that is considered the principle driving force behind leukemic expansion [1].
Amino acids 491 through 876 of BCR constitute a RhoGEF domain that is retained in p210 BCR/ABL1, but not p190 BCR/ABL1 [2]. RhoGEFs activate Rho GTPases, which act as binary switches in the cell [3]. Thus, they interact with their effectors only when GTP-bound. The Rho GTPases are involved in diverse cellular processes, including remodeling of the actin cytoskeleton, cell-cycle progression, morphogenesis, and growth and survival [4]. Accordingly, they are also associated with considerable pathology, including facilitating oncogenic transformation [[5], [6], [7]].
The role of Rho signaling in the hematologic malignancies is currently unclear: While only hyperactivation has been identified in CML [8], both gain- and loss-of-function have been described in other leukemias and lymphomas [[9], [10], [11], [12]]. Accordingly, when several of these alterations were studied in animal models, they variably resulted in both pro- or anti-cancer effects [8,11]. To date, the exploitation of Rho GTPase signaling to treat blood cancers has primarily been via farnesyl transferase inhibition, which disrupts normal Rho GTPase localization to the cell membrane [13]. Their efficacy has varied widely, from significant responses in chronic or advanced-phase CML to no response in older patients with AML [[14], [15], [16], [17], [18]]. Efforts to develop new means of exploiting this pathway are currently underway [19].
Several studies suggest that the RhoGEF domain of p210 BCR/ABL1 may be active and utilize RhoA as a substrate [[20], [21], [22], [23]]. Elevated levels of RhoA-GTP have been observed in cells that express p210 BCR/ABL1, but not p190 BCR/ABL1, or a p210 BCR/ABL1 missense mutation (S509A) that is predicted to eliminate RhoGEF activity [20,22]. When examined in the murine BMT assay for Ph + leukemias, the S509A substitution induces a disease phenotype that is more acute than unmodified p210 BCR/ABL1 and indistinguishable from p190 BCR/ABL1 [22]. This suggests that this domain may account for the different disease outcomes associated with p190 BCR/ABL1 and p210 BCR/ABL1 expression, and that mutations in this domain may trigger disease progression. A recent structural analysis of the isolated RhoGEF domain of BCR suggests that the domain may lack catalytic activity, suggesting that the domain may influence disease progression through a mechanism other than direct engagement of GTPases [24].
Two p210 BCR/ABL1 missense mutations (F547L, T654K) have been previously identified in a CML patient in BC [25]. Although both mutations fall within conserved regions of the RhoGEF domain, their role, if any, in p210 BCR/ABL1-mediated leukemogenesis is currently unknown. To address this, we will evaluate the effects of these substitutions in the murine BMT model for p210 BCR/ABL1-mediated disease. Since it has been shown previously that a p210 BCR/ABL1 mutant that lacks RhoGEF activity causes accelerated disease progression in this model [22], it is possible that these two clinically-derived mutations may have a similar effect, and thus, may account for the more acute disease phenotype that is associated with progression to BC.
Section snippets
Molecular constructs
pAX-bcr(1-876) [26], pAX-bcr-abl1 [21] and the MSCV-IRES-EGFP (MIG) bicistronic retroviral vector (27: Plasmid # 27490, Addgene, Cambridge, MA) have been described previously. MSCV-bcr-abl1/p210-IRES-gfp contains full-length p210 BCR/ABL1 [21]. Using pAX142-bcr(1-876)-HA as a template, the F547L, T654K, and F547L + T654K mutations were introduced via the Quickchange site-directed mutagenesis (SDM) kit according to the manufacturer’s instructions (Stratagene, La Jolla, CA). A unique Fsp1/HindIII
Clinically-derived RhoGEF mutations (CDRMs) do not diminish p210 BCR/ABL1 expression or kinase activity
Substitutions at residues 654 (T654 K) and 547 (F547 L) have been reported in the RhoGEF domain of p210 BCR/ABL1 in a CML patient in BC [25]. In order to characterize the effects of the mutations in the murine BMT model, we introduced them into a retroviral vector containing full-length p210 BCR/ABL1. To ensure that the introduction of the substitutions did not destabilize the resulting proteins, we transiently transfected HEK-293 cells with our retroviral vectors, sorted GFP + cells to
Discussion
We have examined the effects of two clinically derived p210 BCR/ABL1 RhoGEF mutations, alone and in tandem, in a murine BMT model of Ph + leukemia. When compared to mice expressing p210 BCR/ABL1, the CDRMs resulted in a more rapid onset of illness with frequent solid tumors, increased PB disease latency, and they died with an overwhelmingly mature cellular phenotype. These differences indicate that the introduction of the CDRMs altered disease progression and that the mice are dying of a
Declaration of Competing Interest
The authors report no declarations of interest.
Acknowledgement
This work was supported by National Institutes of Health (grant number CA097066)
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