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Page 264 Franca et al. Cancer Drug Resist 2019;2:256-70 I http://dx.doi.org/10.20517/cdr.2019.004

analysis on a panel of 30 human HapMap cell lines trios demonstrated that the intronic SNP rs2413739
(T>C) of PACSIN2 can influence TPMT activity. In ALL patients enrolled in the Total XIIIB/XV protocols
at SJCRH, the TT genotype was associated with lower TPMT activity in patients’ RBC and with an higher
rate of gastrointestinal toxicity during thiopurines therapy. This latter association was also validated in an
[70]
independent cohort of ALL patients treated with the AIEOP-BFM ALL 2000 protocol . A retrospective
study in pediatric ALL patients treated with thiopurines suggested that the presence of rs2413739 in
PACSIN2 gene is a significant risk factor for mercaptopurine induced hematological toxicity in patients with
[71]
wild-type TPMT . It could be hypothesized that the increased toxicities observed in rs2413739 TT carriers
are due to lower TPMT activity. However, no contribution of rs2413739 on thiopurine metabolites (TGN and
MMPN) was reported, thus the mechanistic link between the PACSIN2 SNP and thiopurine adverse effects
is not yet clearly established.

The PACSIN2 SH3 domain can interact with other proteins, such as Ras-related C3 botulinum toxin
substrate 1 (Rac-1) . Several evidences suggest a possible interplay among PACSIN2, Rac-1 and thiopurines.
[67]
The physical interaction between the proline-rich hypervariable region of Rac-1 and the SH3 domain of
PACSIN2 is well known. Due to its role in intracellular vesicle-mediated transport, PACSIN2 could serve as
a molecular brake on Rac-1 signaling, by mediating the Rac-1-GTP internalization toward early endosomes,
[72]
thus slowing down cell processes, such as growth and differentiation, regulated by the small GTPase . The
thiopurine metabolite tGTP binds to Rac-1 as a competitive antagonist of GTP: this binding suppresses the
activation of Rac-1 and leads to apoptosis. Indeed, in IBD patients, effective MP therapy led to decreasing
[73]
concentrations of Rac-1-GTP and Rac-1 expression . PACSIN2 moreover may affect TPMT activity: indeed,
the knock-down of PACSIN2 mRNA in human leukemia cells NALM6 resulted in significantly lower
[70]
TPMT expression and enzymatic activity that can in turn result in a higher tGTP availability and Rac-
1 inhibition. Although not clear enough, these evidences suggest that PACSIN2, Rac-1 and their expression
level could be additional potential biomarkers of thiopurines response and should be further investigated.

Rac-1
[74]
Rac proteins play a major role in T cell development, differentiation, and proliferation and Rac-1 knock-
[75]
outs are embryonic lethal in mice . Rho GTPase proteins cycle between a GDP-bound and a GTP-bound
state through the action of guanine nucleotide exchange factors (GEFs) and the opposing GTPase activating
proteins (GAP); the GTP-bound state is generally considered as the active conformation, regulating signaling
pathways in cells. The binding of tGTP instead of GTP on Rac-1 suppresses its activation, and thus the
activation of its downstream targets such as mitogen-activated protein kinase, NF-κB and the antiapoptotic
protein Bcl-xL. In vivo and in vitro studies suggested that Rac-1 plays a key role in thiopurines mechanism
[24]
of action . Tiede and collaborators have demonstrated that binding of AZA-generated tGTP to Rac-1
induced the mitochondrial pathway of apoptosis in primary CD4+ T lymphocytes lamina propria cells of
[24]
healthy volunteers and CD patients, by downregulating Bcl-xL mRNA and protein . An in vitro study
on primary T cells isolated from healthy donors clarified the molecular mechanism beneath the tGTP-
block of Rac-1, showing that the accumulation of inactive tGDP-Rac-1 proteins over time depends on the
[76]
inhibition of the GEF factor Vav . IBD patients treated with these drugs present a reduced median Rac-
1 expression compared with patients without immunosuppressive therapy, and among MP treated patients,
[73]
non-responders showed an increased median active Rac-1 expression . In animal models, neutrophil and
macrophage conditional Rac1 knock-out show normal bowel histology and do not present any obvious
colonic phenotype; nonetheless they are protected against dextran sulphate sodium-induced colitis and
show a reduced level of the proinflammatory cytokine IL-1β and of the neutrophil chemokine KC compared
[77]
to wild-type mice . The pharmacogenetics of RAC1, a ~30 kDa gene located on p arm of chromosome 7,
has been investigated in IBD, but not in ALL patients. Two intronic RAC1 polymorphisms (rs10951982 and
rs4720672) have been associated with UC susceptibility in a discovery and in 2 independent replication
cohorts; IBD patients who had the rs10951982 G risk allele had increased expression of RAC1 compared