Combined targeted therapy with PI3K and CDK4/6, or FGFR inhibitors show synergistic effects in a neuroblastoma spheroid culture model.

AIM: Neuroblastoma (NB) is, in spite of current intensive therapy with severe side effects, still not cured so new therapies are needed. Recently, we showed combining phosphoinositide 3-kinase (PI3K) (BYL719), fibroblast growth factor receptor (FGFR) (JNJ-42756493) and cyclin-dependent kinase 4/6 (CDK4/6) (PD-0332991) inhibitors, in vitro in NB cell lines grown as monolayers had synergistic effects. However, there were variations depending on the combinations used and the targeted NB cell lines. To obtain further information and to mimic more natural circumstances, we investigated the effects of single and combined administrations of the above inhibitors in spheroid NB-cultures. MATERIAL AND METHODS: Spheroid cultures of NB cell lines SK-N-AS, SK-N-BE(2)-C, SK-N-FI and SK-N-SH were established and treated with single and combined administrations of BYL719, JNJ-42756493, and PD-0332991 and followed for growth, viability, proliferation, cytotoxicity and migration. KEY FINDINGS: Single inhibitor administrations gave dose dependent responses with regard to growth and viability and their combinations were efficient and resulted in a range of additive and synergistic effects. The responses to individual drugs and their various combinations were predominantly alike regardless of whether the cells were cultivated in monolayer or D spheroid NB models. However, in general, slightly higher drug concentrations were necessary in spheroidcultures. SIGNIFICANCE: This study provides pre-clinical evidence that single PI3K, FGFR, and CDK4/6, inhibitors exhibit promising anti-NB activity and when combined lower doses of the drugs could be also used in spheroid NB-cultures, supporting the pursuit of further in vitro and in vivo studies in preparation for future potential clinical use.

Human papillomavirus (HPV) load is higher in HPVDNA/p16 positive than in HPVDNA positive/p16 negative oropharyngeal squamous cell carcinoma but does not differ significantly between various subsites or correlate to survival.

OBJECTIVE: Patients with human papillomavirus DNA positive (HPVDNA+) and p16ink4a overexpressing (p16+) oropharyngeal squamous cell carcinoma (OPSCC), especially those with cancer in the tonsillar and base of tongue subsites as compared to other OPSCC subsites have a better outcome than those with only HPVDNA+ or only p16+ cancer. Likewise having a high viral load has been suggested to be a positive prognostic factor. We therefore hypothesized, that HPV viral load could vary depending on OPSCC subsite, as well as with regard to whether the cancer was HPVDNA+ and p16+, or only HPVDNA+, or only p16+ and that this affected outcome. MATERIAL AND METHODS: To address these issues HPV viral load was determined by HPV digital droplet (dd) PCR in tumor biopsies with previously known HPVDNA/p16 status from 270 OPSCC patients diagnosed 2000-2016 in Stockholm, Sweden. More specifically, of these patients 235 had HPVDNA+/p16+, 10 had HPVDNA+/p16-, 13 had HPVDNA-/p16+ and 12 had HPVDNA-/p16- cancer. RESULTS: We found that HPVDNA+/p16+ OPSCC had a significantly higher viral load than HPVDNA+/p16- OPSCC. Moreover, there was a tendency for a higher viral load in the tonsillar and base of tongue OPSCC subsites compared to the other subsites and for a low viral load to correlate to a better clinical outcome but none of these tendencies reached statistical significance. CONCLUSION: To conclude, the mean viral load in HPVDNA+/p16+ OPSCC was higher than in HPVDNA+/p16- OPSCC, but there was no statistically significant difference in viral load depending on OPSCC subsite or on clinical outcome.

New Approaches Towards Targeted Therapy for Childhood Neuroblastoma.

Neuroblastoma (NB), comprising around 10% of all childhood neoplasms and 15% of pediatric cancer deaths is a heterogenous disease and can be divided into very low-, low-, intermediate- and high-risk NB. Treatment of very low/low-risk NB is usually based on observation, or surgery alone, whereas intermediate-risk NB is in addition to surgery treated with mild chemotherapy and roughly 80-95% of the patients are cured. In contrast, high-risk NB patients receive multimodal therapy with e.g. induction, consolidation, and maintenance therapy, which can include induction chemotherapy and surgery and in severe cases adding intensive chemotherapy and even autologous stem cell transplantation and radiotherapy. Unfortunately, however this treatment does not cure all patients and around 50% succumb to disease. For this purpose, new treatment options are urgently needed. In this review, we describe the complex molecular heterogeneity of NB, and potential new options for targeted therapy.

Efficacy of combined targeted therapy with PI3K and CDK4/6 or PARP and WEE1 inhibitors in neuroblastoma cell lines.

Neuroblastoma (NB), the most frequent solid extracranial tumor in children, is not always cured by current aggressive therapies that have notable adverse effects; therefore, novel treatments are necessary. Phosphoinositide 3­kinase (PI3K) and fibroblast growth factor receptor inhibitors exhibit synergistic effect in NB cell lines. In the present study, mono­ and combination therapy of the United States Food and Drug Administration­approved PI3K, cyclin­dependent kinase­4/6 (CDK4/6), poly­ADP­ribose­polymerase (PARP) and WEE1 G2 checkpoint kinase (WEE1) inhibitors (BYL719, PD­0332991, BMN673 and MK­1775, respectively), were used to treat NB cell lines SK­N­AS, SK­N­BE(2)­C, SK­N­DZ, SK­N­FI and SK­N­SH and viability (assessed by WST­1 assay), proliferation (incucyte analysis) and cell cycle (FACS) changes were assessed. Treatments with all single drugs presented dose­-dependent responses with decreased viability and proliferation and combining BYL719 with PD­0332991 or BMN673 with MK­1775 resulted in additive or synergistic effects in most cell lines., except for SK­N­SH for the former and for SK­N­AS for the latter. Moreover, combining MK­1775 and BMN673 decreased the numbers of cells in S phase to a greater extent than either drug alone, while when combining PD­0332991 and BYL719 the observed effect was close to that of PD­0332991 alone. To summarize, PI3K and CDK4/6 or PARP and WEE1 exhibited synergistic anti­NB effects and lower doses of the inhibitors could be utilized, thereby potentially reducing adverse side effects.

Combination of PARP and WEE1 inhibitors in vitro: Potential for use in the treatment of SHH medulloblastoma.

Medulloblastoma (MB), grouped as either WNT­activated, Sonic hedgehog (SHH)­activated, or non-WNT/non-SHH group 3, accounts for almost 20% of all childhood brain cancers. In spite of current intensive treatments, not all patients are cured and survivors suffer from severe side­effects. The present study therefore examined the effects of the poly­ADP­ribose polymerase (PARP) and WEE1­like protein kinase (WEE1) inhibitors, BMN673 and MK­1775, respectively, alone or in combination on four MB cell lines. More specifically, the MB cell lines, DAOY, UW228­3, MED8A and D425, were tested for their sensitivity to BMN673 and MK­1775 alone or in combination, using cell viability, cell confluency and cytotoxicity assays. The effects on the cell cycle phases were also examined using FACS analysis. Monotherapy with BMN673 and MK­1775 exerted dose­dependent inhibitory effects on the viability of almost all MB cell lines. Notably, when BMN673 and MK­1775 were used in combination, synergistic effects were noted in the SHH group cell lines (DAOY and UW228­3), but not in the already WEE1­sensitive group 3 (MED8A and D425) lines. Moreover, the combination treatment decreased the percentage of cells in the G1 phase and induced the novel distribution of both DAOY and UW228­3 cells in the S and G2/M phases, with the UW228­3 cells exhibiting a greater delay. To conclude, MK­1775 was efficient in all and BMN673 in most cell lines, and their combined use exerted synergistic effects on the SHH, but not the group 3 cell lines. These data suggest that MK­1775 alone may be of interest for all MB cell lines, and that the combination of PARP/WEE1 inhibitors may provide possible therapeutic opportunities for the therapy of SHH MBs. Their use warrants further investigations in the future.

Targeted Therapy of HPV Positive and Negative Tonsillar Squamous Cell Carcinoma Cell Lines Reveals Synergy between CDK4/6, PI3K and Sometimes FGFR Inhibitors, but Rarely between PARP and WEE1 Inhibitors.

Human papillomavirus positive (HPV+) tonsillar and base of tongue squamous cell carcinoma (TSCC/BOTSCC) have a favorable outcome, but upon relapse, survival is poor and new therapeutical options are needed. Recently, we found synergistic effects by combining the food and drug administration approved (FDA) phosphoinositide 3-kinase (PI3K) and fibroblast-growth-factor-receptor (FGFR) inhibitors BYL719 and JNJ-42756493 on TSCC cell lines. Here this approach was extended and Cyclin-Dependent-Kinase-4/6 (CDK4/6) and Poly-ADP-ribose-polymerase (PARP) and WEE1 inhibitors PD-0332991, and MK-1775 respectively were also examined. HPV+ CU-OP-2, -3, -20, and HPV- CU-OP-17 TSCC cell lines were treated with either BYL719 and JNJ-42756493, PD-0332991 BMN-673 and MK-1775 alone or in different combinations. Viability, proliferation, and cytotoxicity were followed by WST-1 assays and the IncuCyte S3 Live® Cell Analysis System. All inhibitors presented dose-dependent inhibitory effects on tested TSCC lines. Synergy was frequently obtained when combining CDK4/6 with PI3K inhibitors, but only sometimes or rarely when combining CDK4/6 with FGFR inhibitors or PARP with WEE1 inhibitors. To conclude, using CDK4/6 with PI3K or FGFR inhibitors, especially PD-0332991 with BYL719 presented synergy and enhanced the decrease of viability considerably, while although dose dependent responses were obtained with PARP and WEE1 inhibitors (BMN-673 and MK-1775 resp.), synergy was rarely disclosed.

Targeted Therapy with PI3K, PARP, and WEE1 Inhibitors and Radiotherapy in HPV Positive and Negative Tonsillar Squamous Cell Carcinoma Cell Lines Reveals Synergy while Effects with APR-246 Are Limited.

Human papillomavirus positive (HPV+) tonsillar and base of tongue cancer (TSCC/BOTSCC) is rising in incidence, but chemoradiotherapy is not curative for all. Therefore, targeted therapy with PI3K (BYL719), PARP (BMN-673), and WEE1 (MK-1775) inhibitors alone or combined was pursued with or without 10 Gy and their effects were analyzed by viability, proliferation, and cytotoxicity assays on the TSCC/BOTSCC cell lines HPV+ UPCI-SCC-154 and HPV- UT-SCC-60A. Effective single drug/10 Gy combinations were validated on additional TSCC lines. Finally, APR-246 was assessed on several TSCC/BOTSCC cell lines. BYL719, BMN-673, and MK-1775 treatments induced dose dependent responses in HPV+ UPCI-SCC-154 and HPV- UT-SCC-60A and when combined with 10 Gy, synergistic effects were disclosed, as was also the case upon validation. Using BYL719/BMN-673, BYL719/MK-1775, or BMN-673/MK-1775 combinations on HPV+ UPCI-SCC-154 and HPV- UT-SCC-60A also induced synergy compared to single drug administrations, but adding 10 Gy to these synergistic drug combinations had no further major effects. Low APR-246 concentrations had limited usefulness. To conclude, synergistic effects were disclosed when complementing single BYL719 BMN-673 and MK-1775 administrations with 10 Gy or when combining the inhibitors, while adding 10 Gy to the latter did not further enhance their already additive/synergistic effects. APR-246 was suboptimal in the present context.

Targeting PI3K, FGFR, CDK4/6 Signaling Pathways Together With Cytostatics and Radiotherapy in Two Medulloblastoma Cell Lines.

OBJECTIVES: Medulloblastoma (MB) is treated with surgery and chemotherapy, with or without irradiation, but unfortunately >20% of the patients are not cured, and treatment comes with serious long-term side effects, so novel treatments are urgently needed. Phosphoinositide 3-kinases (PI3K), fibroblast growth factor receptors (FGFR), and cyclin-D kinases (CDK) play critical roles in cancer, and especially PI3K is crucial in MB, so here targeted therapies against them were explored. METHODS: MB cell lines DAOY and UW228-3 were exposed to PI3K (BYL719), FGFR (JNJ-42756493), and CDK4/6 (PD-0332991) inhibitors, as single or combined treatments, and their viability, cell confluence, apoptosis, and cytotoxicity were examined. Moreover, the inhibitors were combined with cisplatin, vincristine, or irradiation. RESULTS: Single treatments with FGFR, PI3K, or CDK4/6 inhibitors decreased viability and proliferation slightly; however, when combining two inhibitors, or the inhibitors with irradiation, sensitivity was enhanced and lower doses could be used. A more complex pattern was obtained when combining the inhibitors with cisplatin and vincristine. CONCLUSIONS: The data suggest that combination treatments with PI3K, FGFR, and CDK4/6 inhibitors for MB could be beneficial and their use should be pursued further. Likewise, their combination with irradiation gave positive effects, while the addition of cisplatin and vincristine resulted in more complex patterns, which need to be investigated further.

Effects of PI3K and FGFR inhibitors alone and in combination, and with/without cytostatics in childhood neuroblastoma cell lines.

Neuroblastoma (NB) is a heterogenous disease with treatment varying from observation for low­risk tumors, to extensive therapy with chemotherapy, surgery, radiotherapy, and autologous bone­marrow­transplantation and immunotherapy. However, a high frequency of primary­chemo­refractory disease and recurrences urgently require novel treatment strategies. The present study therefore investigated the anti­NB efficacy of the recently FDA­approved phosphoinositide 3­kinase (PI3K) and fibroblast growth factor receptor (FGFR) inhibitors, alpelisib (BYL719) and erdafitinib (JNJ­42756493), alone and in combination with or without cisplatin, vincristine, or doxorubicin on 5 NB cell lines. For this purpose, the NB cell lines, SK­N­AS, SK­N­BE(2)­C, SK­N­DZ, SK­N­FI and SK­N­SH (where SK­N­DZ had a deletion of PIK3C2G and none had FGFR mutations according to the Cancer Program's Dependency Map, although some were chemoresistant), were tested for their sensitivity to FDA­approved inhibitors alone or in combination, or together with cytostatic drugs by viability, cytotoxicity, apoptosis and proliferation assays. The results revealed that monotherapy with alpelisib or erdafitinib resulted in a dose­dependent inhibition of cell viability and proliferation. Notably, the combined use of PI3K and FGFR inhibitors resulted in an enhanced efficacy, while their combined use with the canonical cytotoxic agents, cisplatin, vincristine and doxorubicin, resulted in variable synergistic, additive and antagonistic effects. Collectively, the present study provides pre­clinical evidence that PI3K and FGFR inhibitors exhibit promising anti­NB activity. The data presented herein also indicate that the incorporation of these inhibitors into chemotherapeutic regimens requires careful consideration and further research in order to obtain a beneficial efficacy. Nevertheless, the addition of PI3K and FGFR inhibitors to the treatment arsenal might reduce the occurrence of refractory and relapsing disease in NB without FGFR and PI3K mutations.

Targeting Fibroblast Growth Factor Receptor (FGFR) and Phosphoinositide 3-kinase (PI3K) Signaling Pathways in Medulloblastoma Cell Lines.

BACKGROUND/AIM: Medulloblastoma (MB) accounts for ~20% of pediatric malignant central nervous system tumors. Treatment strategies, including surgery, radiation therapy and/or chemotherapy, are effective, but recurrence and metastasis frequently occur. Therefore, novel therapies are required. Herein, the effects of fibroblast growth factor receptor (FGFR) and phosphoinositide 3-kinase (PI3K) inhibitors on MB cells lines were evaluated. MATERIALS AND METHODS: MB cell lines (UW228-3, DAOY, Med8a, D425, D283) were tested for sensitivity to FGFR (AZD4547) and PI3K (BEZ235 and BYL719) inhibitors by viability, cytotoxicity, apoptosis, and proliferation assays. RESULTS: Single treatments with FGFR and PI3K inhibitors decreased viability and proliferation in a dose-dependent pattern in most cell lines. Combinination of the two type of drugs, increased sensitivity, especially of the most resistant cell line UW228-3. CONCLUSION: Combination treatments with FGFR and PI3K inhibitors were superior to single treatments with FGFR and PI3K inhibitors, especially with BEZ235, for MB cell lines.