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Cordover et al. J Cancer Metastasis Treat 2020;6:45 I http://dx.doi.org/10.20517/2394-4722.2020.101 Page 13 of 19

The group A and group B Paks have approximately 50% amino acid identity in the GBD and kinase
[89]
domains, but differ throughout their other domains . Among the Pak family members, the Pak1 and
[90]
Pak4 genes are located on chromosomal regions that are frequently amplified in cancer . Increased Pak4
levels are associated with oncogenic transformation [91,92] and occur in a number of cancers [93-96] . The wild-
type Pak4 gene was shown to be amplified in a panel of pancreatic cancer samples, in pancreatic ductal
adenocarcinomas [95,97,98] , in squamous cell carcinomas , and in high grade serous and endometrioid
[99]
ovarian tumors [100] . In ovarian cancer cell lines, siRNA studies have shown that Pak4 is one of the genes
whose amplification is critical for cellular viability [100] . High levels of Pak4 are found across all subtypes of
breast cancer including ER+, Her2+, and triple negative [92,101-105] . Thus, Pak4 could serve as a more specific
target for breast cancer therapy.

Pak4 has several cellular functions that may explain why it is so tightly linked with cancer. For example,
Pak4 leads to cytoskeletal changes. This may be directly linked with the control of cell shape, which is
often disrupted in cancer, and with cell migration [106] , a critical function for cancer cells, particularly during
metastasis [Figure 4]. Pak4 has also been shown to have key roles in promoting cell survival [107-109] . It can
promote cell survival and inhibit apoptosis in response to several different stimuli in both fibroblasts and
epithelial cells, including serum withdrawal, or cytokine stimulation, and it can do so by a combination of
different mechanisms, which involve both kinase dependent and kinase independent functions [107-109] . Pak4
can also promote cell proliferation, another important aspect of tumorigenesis, and in some cells, this is
associated with a prolonged activation of the ERK MAP kinase pathway [101] .

Due to the strong link of Pak4 to cancer, several anti-Pak4 compounds have been developed [110-114] . One
of these compounds is KPT-9274. Unlike other Pak4 inhibitors that block PAK4 catalytic activity, KPT-
9274 reduces Pak4 protein levels. This may be important because Pak4 has been shown to have kinase
independent functions [96,108] . For example, a kinase dead Pak4 mutant can inhibit caspase 8 activity and
consequently increase cell survival, as well as a wild-type Pak4. Pak4 can also regulate adhesion turnover
and thereby promote migration, by a mechanism that is independent of its kinase activity [92,113] . KPT-
9274 could thus be more effective in antagonizing Pak4 functions in cancer compared with inhibitors that
specifically block kinase activity. KPT-9274 has shown promise for the treatment of breast cancer, renal
cancer, pancreatic cancer, and others both in vitro and in vivo and is now in a phase I clinical trial for
patients with advanced solid malignancies and non-Hodgkin’s lymphoma (NCT02702492) [114-119] . Although
KPT-9274 was identified as a Pak4 allosteric inhibitor, it also inhibits the metabolic enzyme nicotinamide
phosphoribosyltransferase (NAMPT) by an undefined mechanism [115] . NAMPT is the rate limiting enzyme
for the production of nicotinamide adenine dinucleotide (NAD) via the salvage pathway [120] . NAD is
an important cofactor for redox reactions in metabolic pathways and its levels are tightly controlled by
mechanisms that remain poorly understood. Whether Pak4 is linked to NAMPT, and how dual inhibition
of Pak4 and NAMPT by KPT-9274 leads to growth inhibition of cancer cells warrant further investigation.

Recently, KPT-9274 was not only shown to lead to inhibition of Pak4 and NAMPT in triple negative
breast cancer cells, but also inhibition of Rictor [121] . Rictor is an important component of mTORC2,
Conversely, mouse mammary epithelial cells overexpressing Pak4 had an increase in Rictor activity [121,122] .
AKT phosphorylation at Ser473 was dramatically decreased in response to KPT-9274 treatment, also a
hallmark of mTORC2 activation. These findings reveal that prolonged inhibition of Pak4 by KPT-9274
inhibits mTORC2 activity, and mTORC1 activity was subsequently also found to be decreased. These
results suggest that the anti-proliferative effects of KPT-9274 could be mediated by inhibition of mTOR
signaling. They also raise the possibility that the PAK4 pathway is linked to the mTOR pathway, possibly
via phosphorylation of a component of mTORC2. These results suggest that the simultaneous inhibition of
both Pak4 and mTOR may be an effective way to block the growth of breast cancer cells, and this may have
implications in other types of cancer as well.