Supplementary Table Legends 1-2 from Characterization of a Novel Mitogen-Activated Protein Kinase Kinase 1/2 Inhibitor with a Unique Mechanism of Action for Cancer Therapy

Supplementary Figure 1 from RG7204 (PLX4032), a Selective BRAFV600E Inhibitor, Displays Potent Antitumor Activity in Preclinical Melanoma Models

Supplementary Methods, Tables 1-3, Figure Legend from RG7204 (PLX4032), a Selective BRAFV600E Inhibitor, Displays Potent Antitumor Activity in Preclinical Melanoma Models

Supplementary Table 1 from Characterization of a Novel Mitogen-Activated Protein Kinase Kinase 1/2 Inhibitor with a Unique Mechanism of Action for Cancer Therapy

Supplementary Figures 1-2 from Preclinical In vivo Evaluation of Efficacy, Pharmacokinetics, and Pharmacodynamics of a Novel MEK1/2 Kinase Inhibitor RO5068760 in Multiple Tumor Models

Supplementary Figure 1 from RG7204 (PLX4032), a Selective BRAF^V600E Inhibitor, Displays Potent Antitumor Activity in Preclinical Melanoma Models

PDF file - 311KB, Table S1. Results of proliferation assay. Table S2. Estimated PKPD population parameters. Fig. S1. Schematic view of the PK/PD model. Fig S2. Observed PK parameter and model predicted. Fig S3. Visual predictive check.

Supplementary Figures 1-2 from Identification of the MEK1(F129L) Activating Mutation as a Potential Mechanism of Acquired Resistance to MEK Inhibition in Human Cancers Carrying the B-RafV600E Mutation

Supplementary Methods, Tables 1-3, Figure Legend from RG7204 (PLX4032), a Selective BRAFV600E Inhibitor, Displays Potent Antitumor Activity in Preclinical Melanoma Models

Abstract Purpose: Antitumor clinical activity has been demonstrated for the MDM2 antagonist RG7112, but patient tolerability for the necessary daily dosing was poor. Here, utilizing RG7388, a second-generation nutlin with superior selectivity and potency, we determine the feasibility of intermittent dosing to guide the selection of initial phase I scheduling regimens. Experimental Design: A pharmacokinetic–pharmacodynamic (PKPD) model was developed on the basis of preclinical data to determine alternative dosing schedule requirements for optimal RG7388-induced antitumor activity. This PKPD model was used to investigate the pharmacokinetics of RG7388 linked to the time-course of the antitumor effect in an osteosarcoma xenograft model in mice. These data were used to prospectively predict intermittent and continuous dosing regimens, resulting in tumor stasis in the same model system. Results: RG7388-induced apoptosis was delayed relative to drug exposure with continuous treatment not required. In initial efficacy testing, daily dosing at 30 mg/kg and twice a week dosing at 50 mg/kg of RG7388 were statistically equivalent in our tumor model. In addition, weekly dosing of 50 mg/kg was equivalent to 10 mg/kg given daily. The implementation of modeling and simulation on these data suggested several possible intermittent clinical dosing schedules. Further preclinical analyses confirmed these schedules as viable options. Conclusion: Besides chronic administration, antitumor activity can be achieved with intermittent schedules of RG7388, as predicted through modeling and simulation. These alternative regimens may potentially ameliorate tolerability issues seen with chronic administration of RG7112, while providing clinical benefit. Thus, both weekly (qw) and daily for five days (5 d on/23 off, qd) schedules were selected for RG7388 clinical testing. Clin Cancer Res; 20(14); 3742–52. ©2014 AACR.

As a powerful research tool, siRNA's therapeutic and target validation utility with leukemia cells and long-term gene knockdown is severely restricted by the lack of omnipotent, safe, stable, and convenient delivery. Here, we detail our discovery of siRNA-containing lipid nanoparticles (LNPs) able to effectively transfect several leukemia and difficult-to-transfect adherent cell lines also providing in vivo delivery to mouse spleen and bone marrow tissues through tail-vein administration. We disclose a series of novel structurally related lipids accounting for the superior transfection ability, and reveal a correlation between expression of Caveolins and successful transfection. These LNPs, bearing low toxicity and long stability of >6 months, are ideal for continuous long-term dosing. Our discovery represents the first effective siRNA-containing LNPs for leukemia cells, which not only enables high-throughput siRNA screening with leukemia cells and difficult-to-transfect adherent cells but also paves the way for the development of therapeutic siRNA for leukemia treatment.

494 Background: B-Raf mutations, particularly at V600 occur in 10% of CRCs resulting in constitutive activation of the MAPK pathway. V (RG7204, PLX4032) is a first-in-class, V600B-Raf-selective small molecule inhibitor previously shown to potentiate anti-tumor effects in the V600E CRC xenograft model HT29 in combination (combo) with capecitabine ± bevacizumab (ASCO GI, 2008). V600E CRC respond poorly to EGFR inhibition (EGFRi) both preclinically and clinically. We aimed to determine whether antitumor activity could be potentiated by combining V with EGFRi. Methods: The monotherapy (mono) activities of V, Erl, Erb and Iri were compared to combo of these agents in the HT29 model. V was tested as a mono at its optimal dose (OD), Erl was tested at its MTD or 2/3 MTD, and a combo of V OD + 2/3 MTD Erl were tested. V was subsequently tested as mono or combo at the OD and suboptimal-OD with OD Erb. A final study included Iri as mono, doublets of Iri + V or Erb, and triplet combo of Iri, V, and Erb. Results: Tumor growth inhibition (TGI) and increase in life span (ILS) for combo of OD V + 2/3 MTD Erl was superior to all mono arms. TGI in mono arms was equivalent for V, Erb and Iri. ILS was better for V vs Iri and Erb mono, while Iri vs Erb ILS were equivalent. TGI and ILS for Iri + Erb doublet was equivalent to V mono. Otherwise, TGI and ILS in all other doublets and triplets were superior to mono groups. TGI in V + Erb and V + Iri were equivalent, but ILS was better for V + Iri. TGI and ILS for V + Iri was better than Iri + Erb. TGI in V + Erb was better than Iri + Erb, but ILS was equivalent. TGI in the triplet was superior to all doublets except V + Erb, however ILS was superior to all doublets tested. Conclusions: V potentiates anti-tumor activity in a V600E CRC model when used in combo with Erl, Erb and Iri. Although a V + Erb doublet provided impressive TGI equivalent to a triplet with Iri, the triplet yielded sustained antitumor activity as demonstrated by a significant increase in survival as compared to the doublet. Combo with V may afford V600E CRC patients a differential response to what is seen traditionally with single agent EGFi and is worthy of clinical exploration.

Abstract A high percentage of patients with BRAFV600E mutant melanomas respond to the selective RAF inhibitor vemurafenib (RG7204, PLX4032) but resistance eventually emerges. To better understand the mechanisms of resistance, we used chronic selection to establish BRAFV600E melanoma clones with acquired resistance to vemurafenib. These clones retained the V600E mutation and no second-site mutations were identified in the BRAF coding sequence. Further characterization showed that vemurafenib was not able to inhibit extracellular signal-regulated kinase phosphorylation, suggesting pathway reactivation. Importantly, resistance also correlated with increased levels of RAS-GTP, and sequencing of RAS genes revealed a rare activating mutation in KRAS, resulting in a K117N change in the KRAS protein. Elevated levels of CRAF and phosphorylated AKT were also observed. In addition, combination treatment with vemurafenib and either a MAP/ERK kinase (MEK) inhibitor or an AKT inhibitor synergistically inhibited proliferation of resistant cells. These findings suggest that resistance to BRAFV600E inhibition could occur through several mechanisms, including elevated RAS-GTP levels and increased levels of AKT phosphorylation. Together, our data implicate reactivation of the RAS/RAF pathway by upstream signaling activation as a key mechanism of acquired resistance to vemurafenib, in support of clinical studies in which combination therapy with other targeted agents are being strategized to combat resistance. Cancer Res; 72(4); 969–78. ©2011 AACR.

Hepsin is overexpressed in prostate and other cancers where it is implicated in promoting tumor growth, invasion, and metastasis. To further our understanding of the role of hepsin in prostate and breast cancer, a fully humanized monoclonal antibody that recognizes human and cynomolgus hepsin has been developed. RO5486055 selectively binds to hepsin but not related type II transmembrane serine proteases, and neutralizes hepsin serine protease activity with an IC50 in the single digit nM range. In LNCaP prostate cancer cells, both shRNA knockdown of the hepsin gene and RO5486055 treatment cause a similar accumulation of the β3 chain of laminin-332, a known hepsin substrate. With ip administration in mice, RO5486055 demonstrates dose-dependent exposure and a long serum half-life of 168-406 hours. RO5486055 attenuates tumor growth in the LNCaP prostate cancer and T-47D breast cancer xenograft models, but not in the CRW-22Rv1 prostate or MCF-7 breast cancer xenografts. In these models, it has been shown that the level of the β3 chain of laminin-332 detected by Western blot analysis predicts sensitivity to RO5486055-mediated growth inhibition. Moreover, accumulation of the β3 chain during RO5486055 treatment correlates with antitumor activity. In 8/10 hepsin-expressing patient-derived prostate tumors, an inverse correlation between hepsin and β3 chain expression is observed. The β3 chain of laminin-332 may therefore be useful as both a predictive and response biomarker for anti-hepsin therapy. RO5486055-mediated tumor growth inhibition is enhanced by combination with the EGFR-targeted antibody cetuximab in LNCaP prostate cancer xenografts, and by combination with hormone withdrawal in T-47D breast cancer xenografts. These preclinical results suggest that hepsin-directed therapy could be effective in prostate and breast cancer treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4827. doi:1538-7445.AM2012-4827

Few drugs are currently approved for the treatment of melanoma, including dacarbazine (chemotherapy) and interferon alpha (immunotherapy). However, only a small fraction of patients with metastatic melanoma respond to dacarbazine and a survival benefit has not been shown with this or any other chemotherapy regimen. Taxanes have demonstrated some activity in melanoma, including modest response rates in combination with carboplatin or bevacizumab. These results contrast with the extraordinary Phase I clinical data reported with the BRAFV600E kinase inhibitor RG7204 (PLX4032), which has demonstrated a remarkable response rate (N Engl J Med 363(9):809-819, 2010). Here we report our preclinical investigation of RG7204 in combination with the biologics interferon alpha-2a (Pegasys) or B20-4.1 (B20) (mouse/ human cross-reactive anti-VEGF MAb), and the chemotherapies paclitaxel and carboplatin in the BRAFV600E mutant LOX-IMVI melanoma model in nude mice.The combination of RG7204 and paclitaxel provided superior survival as compared to either monotherapy. Conversely, the combination of RG7204 plus carboplatin or RG7204 plus B20 did not extend survival as compared to RG7204 monotherapy. When RG7204, paclitaxel, and B20 were given in triplet combination, no improvement in survival was observed over any doublet. Similarly, the triplet combination with RG7204, carboplatin and B20 failed to demonstrate improved survival over any doublets or RG7204 monotherapy. Although complete suppression of tumor growth was readily achieved with either RG7204 or Pegasys monotherapy, the combination provided a remarkable survival benefit with median survival extended by nearly a year as compared to either monotherapy. Taken together, these data support clinical testing of RG7204 with interferon alpha in melanoma patients harboring BRAFV600E mutation in order to extend survival, and may allow for use of lower dose interferon. Clinical investigation of RG7204 and paclitaxel combination may also be warranted. While our data do not support combined therapy with RG7204 plus anti-VEGF therapy, it should be noted that the LOX model was resistant to B20 monotherapy, and therefore these findings might not apply to tumors with intrinsic sensitivity to anti-VEGF therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2562. doi:10.1158/1538-7445.AM2011-2562

BACKGROUND:Hormone therapy is the standard of care for newly diagnosed or recurrent prostate cancers. It uses anti-androgen agents, castration, or both to eliminate cancer promoting effect of testicular androgen. The p53 tumor suppressor controls a major pathway that can block cell proliferation or induce apoptosis in response to diverse forms of oncogenic stress. Activation of the p53 pathway in cancer cells expressing wild-type p53 has been proposed as a novel therapeutic strategy and recently developed MDM2 antagonists, the nutlins, have validated this in preclinical models of cancer. The crosstalk between p53 and androgen receptor (AR) signaling suggest that p53 activation could augment antitumor outcome of androgen ablation in prostate cancer. Here, we test this hypothesis in vitro and in vivo using the MDM2 antagonist, nutlin-3 and the p53 wild-type prostate cancer cell line, LNCaP.RESULTS:Using charcoal-stripped serum as a cellular model of androgen deprivation, we show an increased apoptotic effect of p53 activation by nutlin-3a in the androgen-dependent LNCaP cells and to a lesser extent in androgen-independent but responsive 22Rv1 cell line. This effect is due, at least in part, to an enhanced downregulation of AR expression by activated p53. In vivo, androgen deprivation followed by two weeks of nutlin administration in LNCaP-bearing nude mice led to a greater tumor regression and dramatically increased survival.CONCLUSIONS:Since majority of prostate tumors express wild-type p53, its activation by MDM2 antagonists in combination with androgen depletion may offer an efficacious new approach to prostate cancer therapy.

Abstract Although targeting the Ras/Raf/MEK pathway remains a promising anticancer strategy, mitogen-activated protein/extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitors in clinical development are likely to be limited in their ability to produce durable clinical responses due to the emergence of acquired drug resistance. To identify potential mechanisms of such resistance, we established MEK inhibitor–resistant clones of human HT-29 colon cancer cells (HT-29R cells) that harbor the B-RafV600E mutation. HT-29R cells were specifically resistant to MEK inhibition in vitro and in vivo, with drug-induced elevation of MEK/ERK and their downstream targets primarily accountable for drug resistance. We identified MEK1(F129L) mutation as a molecular mechanism responsible for MEK/ERK pathway activation. In an isogenic cell system that extended these findings into other cancer cell lines, the MEK1(F129L) mutant exhibited higher intrinsic kinase activity than wild-type MEK1 [MEK1(WT)], leading to potent activation of ERK and downstream targets. The MEK1(F129L) mutation also strengthened binding to c-Raf, suggesting an underlying mechanism of higher intrinsic kinase activity. Notably, the combined use of Raf and MEK inhibitors overcame the observed drug resistance and exhibited greater synergy in HT-29R cells than the drug-sensitive HT-29 parental cells. Overall, our findings suggested that mutations in MEK1 can lead to acquired resistance in patients treated with MEK inhibitors and that a combined inhibition of Raf and MEK may be potentially useful as a strategy to bypass or prevent drug resistance in the clinic. Cancer Res; 71(16); 5535–45. ©2011 AACR.

8517 Background: Vemurafenib (V) has demonstrated extraordinary results in clinical evaluation, with confirmed response rates of >50%. However, most patients progress eventually, indicating that acquired resistance may impact long-term clinical activity. This study was to elucidate mechanisms of acquired resistance to V in BRAFV600 mutated melanoma in vitro and in vivo. Methods: Six A375 BRAFV600E melanoma clones resistant to V were established by culturing the sensitive cells under continuous V selection to model disease relapse associated with V treatment in melanoma patients. This model system was used in vitro and in vivo to identify potential molecular mechanisms and combination therapies to prevent or mitigate emergence of disease progression. Results: These clones were approximately 100-fold resistant to V in proliferation assays compared to the parental cells, despite retention of the V600E mutation and absence of additional BRAF mutations. High levels of ERK phosphorylation were observed, suggesting that ERK signaling is re-activated in the resistant cells. CRAF protein was upregulated in resistant cells, and knock-down of CRAF restored V-sensitivity in the resistant cells, while overexpression of CRAF in the parental cells conferred resistance to V. Resistance also correlated with increased levels of RAS-GTP, and sequencing of RAS genes revealed an activating mutation in KRAS. High levels of AKT phosphorylation were observed, implicating upregulation of alternative pathways. The combination of V with a MEK inhibitor, but not either compound alone, was able to effectively block ERK activation in resistant cells and to abrogate tumor growth in xenografts, suggesting that ongoing BRAF inhibition is required to block ERK signaling when combined with MEK inhibition. Likewise, V showed strong in vitro synergism with an AKT inhibitor. Conclusions: These data implicate reactivation of the RAS/RAF pathway and activation of an alternative pathway as key mechanisms of disease relapse on V treatment, and support further clinical studies in which a MEK or an AKT inhibitor is added to V treatment to combat resistance.

Few drugs are currently approved for the treatment of melanoma, including dacarbazine (chemotherapy) and interferon alpha (immunotherapy). However, only a small fraction of patients with metastatic melanoma respond to dacarbazine and a survival benefit has not been shown with this or any other chemotherapy regimen. Taxanes have demonstrated some activity in melanoma, including modest response rates in combination with carboplatin or bevacizumab. These results contrast with the extraordinary Phase I clinical data reported with the BRAFV600E kinase inhibitor RG7204 (PLX4032), which has demonstrated a remarkable response rate (N Engl J Med 363(9):809-819, 2010). Here we report our preclinical investigation of RG7204 in combination with the biologics interferon alpha-2a (Pegasys) or B20-4.1 (B20) (mouse/ human cross-reactive anti-VEGF MAb), and the chemotherapies paclitaxel and …