Enhanced synergy of pacritinib with temsirolimus and sunitinib in preclinical renal cell carcinoma model by targeting JAK2/STAT pathway

Abstract

Pacritinib is an oral medication that inhibits several kinases including JAK2, FLT3, IRAK and STAT3. It has been recently approved to treat patients with thrombocytopenia and myelofibrosis. Studies are currently exploring the potential use of pacritinib in treating other types of cancer such as leukaemia, breast cancer and prostate cancer. Our study aimed to investigate the effects of pacritinib alone and its combination with standard of care in renal cell carcinoma (RCC). We showed that pacritinib dose-dependently decreased viability of RCC cells, with IC₅₀ at nanomolar or low micromolar concentration rage. Pacritinib inhibited cell proliferation, decreased colony formation, and increased apoptosis. Interestingly, pacritinib exhibited synergistic effects when combined with temsirolimus and sunitinib, but antagonistic effects when combined with doxorubicin, in a panel of RCC cell lines. We also confirmed that the combination of pacritinib with temsirolimus and sunitinib resulted in synergistic effects in RCC mouse models, with complete inhibition of tumour growth throughout the treatment period. Mechanistic studies indicated that the inhibition of JAK2, but not IRAK, was the main contributor to the anti-RCC activity of pacritinib. Our study is the first to demonstrate that pacritinib shows promise as a treatment option for RCC and underscores the therapeutic potential of targeting the JAK2/STAT signalling pathway in RCC.

Keywords:

• Pacritinib
• temsirolimus
• doxorubicin
• sunitinib
• JAK2/STAT
• RCC

Introduction

Renal cell carcinoma (RCC) is a prevalent urinary system malignancy with a rising incidence globally [1]. Despite notable strides in cancer genomics and the advancement of innovative pharmacological treatments, effectively managing renal cell carcinoma remains a considerable challenge. Radical nephrectomy stands as the primary treatment for early-stage RCC patients; however, over 30% of RCC cases are diagnosed at an advanced/metastatic stage [2]. Patients with distant metastatic disease have a 5-year survival rate of <10% [3]. Clear cell renal cell carcinoma (ccRCC), the most prevalent type of kidney cancer, is characterized by VHL gene mutation and the consequent activation of hypoxia-inducible factor-2α (HIF-2α), which results in the overexpression of vascular endothelial growth factor (VEGF) and a pro-angiogenic microenvironment [4]. Antiangiogenic agents targeting the VEGF pathway, such as tyrosine kinase inhibitors (TKIs) sunitinib and mTOR inhibitors temsirolimus and everolimus, have shown some improvement in clinical outcomes for metastatic ccRCC [5, 6]. However, such treatments rarely achieve a complete response and are not curative [7]. Recent findings have demonstrated that the anthracycline chemotherapy drug, doxorubicin, which inhibits topoisomerase, exhibits clinical effectiveness when used in combination with gemcitabine for patients with platinum-refractory RCC [8]. There is a need to identify more effective treatment strategies for RCC.

The role of the JAK/STAT axis in tumorigenesis and cancer development is highly significant [9]. Within this axis, STAT3 functions as an oncogenic transcription factor, promoting the expression of genes associated with proliferation, anti-apoptosis, survival, and metastasis. The observed overactivation of STAT3 in numerous human malignancies, including gastrointestinal tumours, drives tumour progression, metastasis, and contributes to drug resistance [10]. Notably, recent research has shed light on the regulatory importance of noncoding RNAs (ncRNAs), encompassing microRNAs, long noncoding RNAs, and circular RNAs, in modulating the JAK/STAT pathway, further impacting its oncogenic potential [11, 12].

Pacritinib is an oral kinase inhibitor that can target wildtype Janus associated kinase 2 (JAK2), mutant JAK2^V617F, ACVR1 (ALK2), IRAK1 and FMS-like receptor tyrosine kinase 3 (FLT3). These proteins are involved in the signalling of several cytokines and growth factors that are important for haematopoiesis, immune function, and cancer [13, 14]. Pacritinib therapy was well tolerated and induced significant and sustained spleen volume reduction and symptom alleviation in patients with myelofibrosis. Most patients experienced hematologic stability and low-grade GI toxicities, with diarrhea and nausea representing the most common nonhematologic adverse effects [15–17]. Additionally, preclinical studies and a pilot phase I study have shown that pacritinib is well-tolerated and exhibits anti-leukemic activity when used in combination with chemotherapy in patients with FLT3 mutations [18]. Ongoing trials (Trail No. NCT04520269 and NCT04635059) are evaluating the potential of pacritinib in breast and prostate cancer are ongoing, while preclinical research suggests its potential efficacy against glioblastoma, non-small cell lung cancer, breast cancer, and nasopharyngeal carcinoma [19–22]. However, little is known about pacritinib’s effectiveness in treating RCC.

In the present study, we aimed to assess the effects of pacritinib as single drug by utilizing a panel of RCC models. We also examined whether pacritinib could increase the effectiveness of chemotherapy or targeted therapy, such as temsirolimus, sunitinib, and doxorubicin. We validated the in vitro results through the use of a mouse model of RCC. Additionally, we investigated the underlying mechanisms behind pacritinib’s activity in RCC.

Materials and methods

Cell lines, drugs and treatment

The cell lines used in this study were either obtained from the Cell Bank of Type Culture Collection of Chinese Academy of Sciences (A-498, Caki-1, RCC4, and ACHN) or the American Type Culture Collection (786-O, Caki-2, and 769-P). The cells were cultured in Eagle’s Minimum Essential Medium (EMEM) supplemented with 10% fetal bovine serum and Penicillin-Streptomycin under a 5% CO2 atmosphere. All culturing reagents were procured from Invitrogen. Sunitinib (Cat No. S7781), temsirolimus (Cat No. S1044), pacritinib (Cat No. S8057), and doxorubicin (Cat No. S1208) were obtained from Selleckchem, and the purity of these chemical compounds was determined to be >97% based on the manufacturer’s HPLC data. Specific treatment conditions for each drug were indicated in the figure legends. In all the in vitro assays, drug treatment was administered using the culture medium.

2.2. Cell viability assay and quantitative analysis of drug interactions

After 24 h of drug treatment, the cell viability was evaluated using the MTT assay kit (Abcam), following the manufacturer’s protocol. The Prism software was used to determine the half maximal inhibitory concentration (IC₅₀) of each drug. For combination studies, the cells were treated with a single drug or combination at an equipotent constant-ratio concentration of their IC₅₀ multiplicity. The CalcuSyn software was used to perform the combination index (CI) analysis. CI values of less than 1 were considered to indicate synergistic effects, CI values between 1 and 1.1 were considered to indicate additive effects, and CI values greater than 1.1 were considered to indicate antagonistic effects [23].

Cell proliferation and apoptosis assays

Cell proliferation was evaluated using the BrdU proliferation assay kit (Abcam), while apoptosis was determined using the cell death detection ELISA kit (Roche). 10⁴ cells were seeded onto 96-welll plate for proliferation assay and 10⁶ cells were seeded onto 6-well plate for apoptosis assay. Both procedures were performed strictly following the manufacturer’s protocol. The absorbance was measured using a Tecan plate reader.

Soft agar colony formation assay

Cells were seeded at a density of 10⁵ cells/well in 6-well plates with 0.35% noble agar (Sigma) mixed with EMEM on top of 0.5% noble agar with medium. The colonies were then incubated for 10–14 days. The resulting colonies were fixed using 6.0% v/v glutaraldehyde and stained with 0.5% w/v crystal violet. The images of the plates were captured using a stereomicroscope.

Western blotting

The primary antibodies used in this study for Western blotting were p-JAK2 (Cat No. 3771), JAK2 (Cat No. 3773), p-STAT3 (Cat No. 9131), STAT3 (Cat No. 9132), p-STAT5 (Cat No. 9359), STAT5 (Cat No. 9363), p-IRAK (Cat No. ab218130), IRAK (Cat No. 4504), p-Akt (Cat No. 9271), Akt (Cat No. 9272), p-Erk (Cat No. 9101), and Erk (Cat No. 9102), which were obtained from Cell Signalling Technology. A total of 2 x 10⁶ cells were seeded onto a 6-well plate and treated with pacritinib for 24 h. After the treatment, the cells were lysed using RIPA buffer (Invitrogen) to extract total proteins, and the protein concentration was determined using the BCA protein assay kit (Abcam). Subsequently, 40 µg of protein from each sample was loaded onto an SDS-PAGE gel and resolved through electrophoresis. The proteins were then transferred to nitrocellulose membrane and incubated with primary antibodies overnight at 4 °C, followed by 2 h of incubation with the secondary antibodies. Finally, protein visualization was accomplished using enhanced chemiluminescence, following the manufacturer’s instructions (Amersham).

DNA transfection

Transfections were performed in 6-well plates by adding 1 µg of vector control and JAK2-overexpressing plasmid along with DharmaFECT transfection reagent to the culture medium when the cells reached 80% confluence, followed by incubation for 12 h. The full-length coding sequence of JAK2 was amplified from normal cDNA using Platinum PCR SuperMix High Fidelity (Invitrogen) and then cloned into the pCMV3-C-his vector (Sino Biological). After 48 h post-transfection, the medium was replaced with fresh culture medium and the JAK2 protein level was examined.

RCC models in SCID mouse

SCID mice (Biocytogen Inc) were used for the study, and they were injected subcutaneously with 5 × 10⁶ Caki-1 cells in 100 µl serum-free medium. Once the tumours had developed successfully, the mice were divided into 8 groups and treated differently, as specified in the figure legends, with varying doses and durations. Temsirolimus and doxorubicin were given via intraperitoneal injection, while sunitinib and pacritinib were administered via oral gavage. The animals’ drug toxicity was monitored during the treatment period. Sunitinib and pacritinib were formulated using 0.5% methyl cellulose and 1% Tween-80 in ddH₂O, while doxorubicin and temsirolimus were prepared with 5% DMSO, 40% PEG 300, and 5% Tween80 in ddH₂O. These solutions served as the vehicle for each drug. All drugs were freshly prepared before use. All drugs were freshly prepared before use. The tumour volumes were calculated using the formula (length)2 x (width)/2. The Clarke’s CI (CCI) Equation was used to determine the combination effects in vivo: CCI = A/B – (C/B × D/B), where A represents the average tumour measurement from the combination group; B represents the average measurement from the vehicle control; and C and D represent the average measurements from monotherapy 1 and 2, respectively [24]. A CCI value of < 0 is considered a synergistic effect.

Statistical analyses

The in vitro assay results were obtained by performing at least three independent experiments, each comprising duplicate or triplicate samples. The data underwent analysis using a one-way analysis of variance, followed by post hoc comparisons using the Statistical Package for the Social Sciences. Statistical significance was determined using two-tailed tests, and discrepancies were considered statistically significant if the P value was equal to or below 0.05.

Results

Pacritinib displays potent inhibitory effects in RCC cells

In this study, we chose a diverse panel of renal cell carcinoma (RCC) cell lines, namely Caki-2, ACHN, Caki-2, A498, 786-O, 769-P, and RCC4, to effectively demonstrate the biological effects of pacritinib. These selected cell lines represent both primary and metastatic clear cell and papillary RCC, each characterized by different molecular backgrounds [25]. Specifically, Caki-1 represents metastatic clear cell RCC, RCC-4, and 786-O represent primary clear cell RCC, ACHN represents metastatic papillary RCC, and Caki-2 represents primary papillary RCC. Moreover, 786-O, 769-P, and RCC4 harbour VHL mutations, while A-498 and Caki-1 harbour SETD2 mutations. Cells were treated with different concentrations of pacritinib for 24 h, and the level of metabolic activity was measured to indicate cell viability, proliferation and cytotoxicity. As shown in Figure 1A, pacritinib treatment inhibited RCC cell growth in a dose-dependent manner in all tested cell lines. The IC₅₀ values of pacritinib ranged from 0.28 to 7.6 µM, with Caki-2 being the least sensitive and Caki-1 being the most sensitive to pacritinib treatment. We also determined the IC₅₀ values of RCC drugs, including doxorubicin, temsirolimus and sunitinib, and found that the efficacy of pacritinib was comparable or even better than these drugs (Figure 1B). Specifically, the IC₅₀ ranges of doxorubicin, temsirolimus and sunitinib were 2.3 to 10.2 µM, 8.5 to 24.3 µM and 3.6 to 18.2 µM, respectively.

Figure 1. Pacritinib displays potent anti-RCC activity in a panel of RCC cell lines. (A) Pacritinib dose-dependently decreases RCC cell viability. Results were presented as relative to control (value set as 1). (B) IC₅₀ values calculated from the results of the MTT assay. Cells were treated with test compound for 24 h. Experiments were repeated at least three times to derive averages. Data are presented as mean ± SD.

Pacritinib synergizes with temsirolimus and sunitinib but not doxorubicin in RCC cells

We next investigated the combinatory effect of pacritinib with temsirolimus, sunitinib and doxorubicin using the ‘Chou-Talalay’ method and MTT assay [26]. We combined pacritinib with each drug at a ratio based on their IC₅₀ values and found that the CI value of the pacritinib and temsirolimus combination was <1 in Caki-2, ACHN, Caki-1, A498, and RCC4 cell lines, ∼1 in 786-O, and >1.1 in 769-P (Figure 2A). This indicates that pacritinib and temsirolimus combination is synergistic, additive and antagonist in 5, 1 and 1 RCC cell lines, respectively. Similarly, the CI value of pacritinib and sunitinib combination was <1 in Caki-2, ACHN, Caki-1, A498, and 769-P, ∼1 in RCC4, and >1.1 in 786-O. However, the CI value of pacritinib and doxorubicin was <1 in 786-O and RCC4, ∼1 in ACHN, and >1.1 in Caki-2, Caki-1, A498, and 769-P. Our combination analysis showed that pacritinib synergizes with temsirolimus and sunitinib but antagonizes with doxorubicin in the majority of RCC cell lines. This result was consistent with our colony formation assay, where the combination of pacritinib with temsirolimus or sunitinib decreased the number of colonies more than a single drug alone (Figure 2B). However, the combination of pacritinib with doxorubicin did not decrease the number of colonies further.

Figure 2. Pacritinib synergizes with sunitinib and temsirolimus in inhibiting RCC in vitro. (A) Combination index (CI) values calculated for various test compound combinations using CalcuSyn. CI values were generated by non-linear regression methods. Synergistic combinations are highlighted in red. Cells were treated with test compound for 24 h. (B) Representative images showing colony formation of Caki-1 cells in the presence of various test compound combinations. Pacritinib at 0.5 µM, temsirolimus at 5 µM, sunitinib at 4 µM and doxorubicin at 5 µM were used. Experiments were repeated at least three times to derive averages. Data are presented as mean ± SD.

Pacritinib inhibits proliferation, anchorage-independent colony formation and survival in RCC cells

We conducted experiments to investigate the effects of pacritinib on various biological activities in RCC cells. Specifically, we evaluated its impact on cell growth, anchorage-independent colony formation, and apoptosis. Using a BrdU incorporation assay, we observed that pacritinib caused a significant decrease in cell proliferation in Caki-1 and ACHN cells (Figure 3A). We also observed a significant reduction in anchorage-independent growth as measured by soft agar colony formation (Figure 3B). Furthermore, pacritinib induced apoptosis in RCC cells as evidenced by an increase in DNA fragmentation (Figure 3C). Notably, we observed that the effectiveness of pacritinib in inhibiting cell growth and inducing apoptosis was dependent on the dose administered.

Figure 3. The inhibitory effects of pacritinib on the multiple biological activities of RCC cells. Pacritinib significantly decreases proliferation (a), inhibits colony formation (B) and increases apoptosis (C) in Caki-1 and ACHN cells. Proliferation and apoptosis were evaluated after 72 h drug treatment. *, p < 0.05; **, p < 0.01; ***, p < 0.001 compared to control. Experiments were repeated at least three times to derive averages and results were presented as relative to control (value set as 1). data are presented as mean ± SD.

Pacritinib acts on RCC cells in a JAK2-dependent manner

Studies have shown that the anti-cancer activities of pacritinib are attributed to its enzyme inhibitory properties on JAK2, STAT3 and IRAK [19, 22]. We performed immunoblotting analysis of these molecules in Caki-1 cells after pacritinib treatment. We showed that effective concentrations of pacritinib significantly reduced phosphorylation of JAK2 and STAT3 in RCC cells (Figure 4A and B). Consistent with previous reports [20, 27], pacritinib also decreased phosphorylation of STAT5, Akt and Erk (Figure 4A and B). However, IRAK phosphorylation was not affected by pacritinib treatment. Furthermore, ectopic expression of JAK2 partially but significantly reversed the anti-proliferative and pro-apoptotic effects of pacritinib in Caki-1 cells (Figure 4C and D, and Figure S1), confirming that pacritinib’s effects on RCC cells are at least partially dependent on JAK2. Of note, the decreased phosphorylation of JAK2, STAT3, Akt and Erk induced by pacritinib treatment and the reverse of pacritinib’s inhibitory by JAK2 overexpression are not limited to Caki-1 cells; ACHN cells responded in a similar manner (Figure S2), suggesting that JAK2 inhibition is a general underlying mechanism of pacritinib’s action in RCC cells. We further found that ruxilotinib, a specific and potent JAK1 and JAK2 inhibitor [28], inhibited phosphorylation of JAK2 but not IRAK1 in ACHN and Caki-1 cells (Figure S3A). In addition, the combination of ruxolitinib and temsirolimus is synergistic in RCC cells (Figure S3B). Collectively, our results clearly demonstrate that JAK2 inhibition is the underlying mechanism of pacritinib’s action in RCC cells.

Figure 4. Pacritinib acts on RCC in JAK2-dependent manner. (A and B) Representative images and quantification of Western blotting of p-JAK2, p-STAT3, p-STAT5, p-Akt and p-Erk in Caki-1 cells after 24 h treatment of pacritinib. Quantification of band density was performed using Image J. Overexpression of JAK2 significantly reversed the anti-proliferative (C) and pro-apoptotic (D) effects of pacritinib in Caki-1 cells. Pacritinib at 5 µM was used in proliferation and apoptosis assays. The evaluation of proliferation involved the use of the BrdU assay, which measures BrdU incorporation by proliferating cells. To assess apoptosis, the cell death detection ELISA kit was employed, which measures DNA fragmentation occurring in apoptotic cells. *, p < 0.05; **, p < 0.01, compared to vector.

Pacritinib synergizes with temsirolimus and sunitinib but not doxorubicin in RCC model in mice

We selected the Caki-1 model of RCC to investigate the in vivo combination of pacritinib with temsirolimus, sunitinib, or doxorubicin because this cell line is a widely used model for metastatic ccRCC [25]. We only initialized drug treatment when tumour reached ∼200 mm³. In the control group, tumour growth was slow during the first two weeks but accelerated during the third and fourth weeks, with tumours reaching approximately 1500 mm³ by the end of week 4. As a monotherapy, all tested drugs significantly reduced tumour growth compared to the control group (Figure 5). Treatment with pacritinib at 100 mg/kg (o.b.) once daily, temsirolimus at 2 mg/kg (i.p.) once daily, sunitinib at 40 mg/kg (o.b.) once daily, and doxorubicin at 5 mg/kg (i.p.) once weekly resulted in approximately 30%, 60%, 50%, and 45% inhibition of tumour growth by week 4, respectively. However, tumours in all groups receiving monotherapy continued to grow and eventually reached similar sizes to those in the control group after 1 to 3 weeks.

Figure 5. Pacritinib combined with sunitinib or temsirolimus is efficacious and synergistic in inhibiting Caki-1 growth in mice. In combination groups, both single drugs were administrated in half the volume. Mice with tumour size ∼1600 mm³ were euthanized. Mice SCID mice (n = 10 per group) were inoculated with Caki-1 cells. Tumour volume was measured at the indicated timepoints. Sunitinib at 40 mg/kg once daily; temsirolimus at 2 mg/kg once daily; pacritinib at 100 mg/kg once daily and doxorubicin at 5 mg/kg once weekly. ***, p < 0.001, compared to single drug alone.

The combination of pacritinib and temsirolimus completely halted tumour growth for up to 8 weeks. When pacritinib was combined with sunitinib, tumour growth was inhibited by approximately 90% and 60% by week 4 and week 8, respectively, with a CCI value of −0.08 indicating synergy. However, the combination of pacritinib and doxorubicin did not show improved efficacy compared to single drug treatment. No noticeable toxicity was observed in any treatment group up to week 4, indicating good tolerance in mice.

To exclude the possibility that the synergy is cell line-dependent, we generated an in vivo RCC model using Caki-2 cells, which are less sensitive to pacritinib and sunitinib compared to Caki-1. We investigated the efficacy of pacritinib and sunitinib alone and in combination. Our findings revealed that pacritinib and sunitinib moderately inhibited Caki-2 growth when administered individually (Figure 6A). However, when used in combination, they remarkably inhibited Caki-2 growth in vivo. This observation aligns with our in vitro results that demonstrated a synergistic effect of pacritinib and sunitinib in Caki-2 cells.

Figure 6. Pacritinib combined with sunitinib is efficacious and synergistic in inhibiting Caki-2 growth in mice. (A) Caki-2 growth curve in mice treated with pacritinib, sunitinib and the combination. In combination groups, both single drugs were administrated in half the volume. Mice with tumour size ∼1600 mm³ were euthanized. Mice SCID mice (n = 5 per group) were inoculated with Caki-2 cells. Tumour volume was measured at the indicated timepoints. Sunitinib at 40 mg/kg once daily; pacritinib at 100 mg/kg once daily. (B and C) Western blotting of p-JAK2 and p-VEGFR2 of tumour lysates. Tumour lysates from each treatment group were pooled for immunoblotting. Quantification of band density was performed using Image J. ***, p < 0.001, compared to single drug alone.

Furthermore, through immunoblotting analysis, we observed decreased levels of p-JAK2 and p-STAT3 in the pacritinib group, reduced p-STAT3 and p-VEGFR2 in the sunitinib group, and decreased p-JAK2, p-STAT3, and p-VEGFR2 in the combination group (Figure 6B). These results indicate that the synergistic action of pacritinib and sunitinib can be attributed to the dual inhibition of JAK2- and VEGF-mediated signalling pathways.

Discussion

Targeting HIF2-VEGF axis and PI3K/Akt/mTOR signalling pathway which are hyperactive in RCC has shown initial clinical responses but is not sustained over the long term as patients developed resistance [29]. One of the most common strategies to reverse or overcome drug TKI resistance is the use of combinations of agents with different mechanisms of action and molecular targets. Since the identification of JAK mutations in many cancers, the JAK/STAT signalling pathway has been shown to play important roles in tumorigenesis, maintenance and metastasis of breast cancer, leukaemia, stomach and lung cancer [30, 31]. To our knowledge, this study demonstrates for the first time that inhibition of JAK/STAT synergizes preferentially with temsirolimus and sunitinib in preclinical RCC models, and this can be achieved with pacritinib for translational purpose in clinics.

Earlier studies have demonstrated that pacritinib exhibits activity against FLT3-ITD + leukaemia primary cells and non-small cell lung cancer resistant to EGFR-TKI treatment [20, 32]. We demonstrate that pacritinib possesses potent anti-RCC activity, with clinically achievable IC₅₀, regardless of the cellular origin and genetics of the RCC. Additionally, pacritinib’s inhibitory effect on anchorage-independent colony formation suggests that it is effective against subpopulations of RCC stem-like cells and cells with metastatic potential [33, 34]. This observation is further supported by Jensen et al.’s work that found pacritinib to be effective in inhibiting tumour-initiating cells derived from glioblastoma patients [19].

The lack of synergy between pacritinib and doxorubicin in RCC, in contrast to the observed synergies with temsirolimus and sunitinib, indicates that pacritinib’s enhancement of these drugs’ efficacy is not due to its inhibition of P-glycoprotein (P-gp) activity. Unlike fedratinib, another JAK2 inhibitor that inhibits P-gp activity and induces cytotoxicity in antimitotic drug-treated P-gp overexpressing resistant cancer cells [35], our mechanism studies demonstrated that pacritinib’s action in RCC is attributable to JAK2/STAT inhibition. Adrian et al. reported a JAK2/STAT5-triggered positive feedback loop that reduces the efficacy of PI3K/mTOR inhibition and showed that JAK2 inhibition eliminates this feedback loop, leading to the synergistic inhibition of cancer with combined PI3K/mTOR and JAK2 inhibition [36]. Temsirolimus and sunitinib have shown some improvement in clinical outcomes for metastatic RCC through inhibiting mTOR signalling and pro-angiogenic signalling, respectively [5, 6]. Pacritinib’s unique feature lies in its capacity to inhibit JAK/STAT signalling, which plays a crucial role in cancer growth and progression. By combining pacritinib with temsirolimus and sunitinib, we speculate that the dual inhibition of JAK/STAT and mTOR or VEGF pathways may lead to a synergistic effect, enhancing the overall therapeutic outcome in the context of metastatic RCC (Figure 7). The differences in the combinatory effects of pacritinib with temsirolimus and sunitinib in various cell lines suggest that the combination is influenced by diverse cellular origins and genetic profiling. Various studies have consistently demonstrated inhibition of IRAK1 phosphorylation in both leukaemia cells and macrophages [37, 38]. However, our observations show that pacritinib does not inhibit IRAK1 phosphorylation in RCC cells. While it has been established that pacritinib exhibits a high-affinity binding to the kinase pocket of mutant IRAK1, it is worth noting that the sensitivity to pacritinib is diminished in cases where mutant IRAK1 is present, particularly with the D298K substitution, which confers a significant reduction in pacritinib sensitivity [37]. Among the various factors that could potentially influence pacritinib’s effectiveness against IRAK1, including kinase pocket binding, binding kinetics, cellular environment, and post-translational modifications of IRAK1, we hypothesize that IRAK1 mutations may play a pivotal role. Therefore, investigating the potential impact of IRAK1 mutations within the context of RCC presents an intriguing avenue for exploration.

Figure 7. The flow chart depicting the underlying mechanisms of synergism by pacritinib in combination with sunitinib or temsirolimus in RCC.

Our data obtained from RCC mouse model confirm the in vitro findings and are of significant value. In mice with well-established and aggressive Caki-1 tumour, pacritinib at non-toxic dose started to inhibit tumour growth after 7 days of dosing. While there was a significant inhibition of tumour growth in the pacritinib-treated group, tumours continued to progress in the standard of care groups. In contrast, the combination of pacritinib with temsirolimus and sunitinib, but not doxorubicin, was found to be synergistic in inhibiting tumour growth. However, a slow but firm tumour progression was observed in the pacritinib and sunitinib combination group but not in the pacritinib and temsirolimus combination group, starting from week 6, suggesting the development of resistance to pacritinib and sunitinib.

Previous studies have reported that pacritinib can improve overall median survival in combination with temozolomide in mice with an aggressive recurrent glioblastoma [19]. The combination of pacritinib with SMO inhibitors suppresses lung and liver metastases, and prolongs host survival without toxicity in mice with intracardially inoculated triple negative breast cancer cells [21]. Although there is no objective responses in a single arm trial of pacritinib in refractory metastatic colon and rectal cancer [39], novel combinations with pacritinib could be explored. Our study, along with numerous other studies on the combinatory effects of pacritinib with anti-cancer agents in various cancer models in mice, provides compelling evidence to support the clinical trials aimed at evaluating pacritinib’s potential for treating cancer patients.

In conclusion, our study shows that pacritinib has potential as a treatment option for advanced RCC due to its ability to synergize with standard of care drugs in RCC models, as well as its favourable pharmacokinetic and safety profile.

Authors’ contributions

LL and YT designed and supervised the study, FM and LG performed the experiments, FM, LG and LL interpreted the data, YT wrote the manuscript. All authors revised the manuscript and approved the final version.

Informed consent

Not applicable

Research involving human participants and/or animals

The Institutional Animal Care and Use Committee of Xiangyang No.1 People’s Hospital granted approval for this study (Approval number XYYYE20220099). All procedures conducted adhered to the ethical standards set forth by the 1964 Helsinki declaration and its subsequent amendments, or equivalent ethical guidelines.

Availability of data and materials

Data available within the article.

Disclosure statement

No potential conflict of interest was reported by the author(s)

Additional information

Funding

This work was supported by a research grant provided by the Health and Family Planning Commission of Hubei Province (WJ2017M232).