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Integrated proteomic and metabolic single-cell assays reveal multiple independent adaptive responses to drug tolerance in a BRAF-mutant melanoma cell line Cancers commonly develop resistance against chemotherapeutics or targeted therapies through various types of genetic or non-genetic mechanisms. Non-genetic mechanisms have been shown to occur early on and can provide a latent reservoir of cells for the emergence of various different type of mechanisms, yet very limited understanding of process were resolve main from bulk analysis. Considering the heterogeneous nature of the tumor cells, a single-cell level characterization of the process worth detailed further investigation. Using MAPK inhibition of BRAF-mutant melanomas as a model system, we resolved that cells take different paths to go from drug-sensitive to drug-resistant state. Using a microfludic-based single-cell integrated proteomic and metabolic assay, we assayed for a panel of signaling, phenotypic, and metabolic regulators at four time points during the first five days of drug treatment. Dimensional reduction of the resultant data set, coupled with information theoretic analysis, uncovered a complex cell state landscape and identified two distinct paths connecting drug-naïve and drug-tolerant states. Cells are shown to exclusively traverse one of the two pathways depending on the level of the lineage restricted transcription factor MITF in the drug-naïve cells. The two trajectories are associated with distinct signaling and metabolic susceptibilities, and are independently druggable. Our results update the paradigm of adaptive resistance development in an isogenic cell population and offer insight into the design of more effective combination therapies. Citation Format: Yapeng Su, Guideng Li, Melissa Ko, Hanjun Cheng, Ronghui Zhu, Min Xue, Lidia Robert, Raphael Levine, Antoni Ribas, Garry Nolan, Wei Wei, Sylvia Plevritis, David Baltimore, James R. Heath. Systems biology for investigating drug resistance mechanism of melanoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6585.

The treatment landscape for metastatic melanoma has changed considerably over the last several years with the approval of targeted therapy, such as BRAF inhibitors, and immunotherapy, such as immune checkpoint inhibitors that block cytotoxic T lymphocyte associated protein 4 (CTLA-4) and programmed cell death 1 (PD-1). However, approximately 40% of patients do not respond to the current standard of care, and up to one-third of patients who initially respond to therapy can relapse. Novel therapies that act by a distinct mechanism of action may be needed to increase durable response rates in metastatic melanoma. Bispecific T cell engager (BiTE®) antibody constructs directly connect T cells to tumor cells and redirect T cell lysis to achieve tumor cell killing. BiTE®-mediated cytotoxicity is not dependent on a specific T cell receptor or peptide-MHC complex and has the potential to circumvent common immune resistance mechanisms. We have generated a BiTE® with half-life extension (HLE) that targets the atypical cadherin CDH19 on melanoma cells and CD3 on T cells (anti-CDH19/CD3 HLE BiTE®). We have also generated an HLE BiTE® that targets delta-like ligand 3 (DLL3) on tumor cells and CD3 on T cells, anti-DLL3/CD3 HLE BiTE®, or AMG 757. CDH19 and DLL3 are expressed on about half of primary and metastatic melanoma tumors prior to treatment. In this study, we investigated the expression of CDH19 and DLL3 and the activity of the corresponding BiTE® antibody constructs in a panel of 14 melanoma cell lines that were derived from patients that were resistant to either targeted therapy or immune checkpoint therapy and expanded the spectrum of recently defined multistage differentiation melanoma subtypes from melanocytic, transitional, neural crest-like and de-differentiated subtypes (Tsoi et al. Cancer Cell 2017). The cell surface expression of CDH19 and DLL3 was determined by flow cytometry. We then used T cell-dependent cytotoxicity (TDCC) assays to assess the activity of the anti-CDH19/CD3 HLE BiTE® and AMG 757 against the cell lines. 13/14 (93%) of the treatment-resistant melanoma cell lines expressed CDH19 on the cell surface, and 9/14 (64%) of the cell lines expressed cell surface DLL3. The anti-CDH19/CD3 HLE BiTE® and AMG 757 demonstrated potent cell killing of CDH19-positive or DLL3-positive melanoma cells, respectively, in TDCC assays, with EC50 values in the low picomolar range. These data show that melanoma cells that become resistant to targeted therapy and/or immune checkpoint blockade can be effectively targeted with BiTE® antibody constructs and suggest that the anti-CDH19/CD3 HLE BiTE® and AMG 757 may be potential novel therapies for treatment of relapsed and/or refractory metastatic melanoma. Citation Format: Julie M. Bailis, Fei Lee, Michael Giffin, Paul Hughes, Jennifer Tsoi, Lidia Robert, Thomas G. Graeber, Antoni Ribas, Angela Coxon. Melanoma subtypes that emerge during adaptive resistance to therapy are targets for bispecific T cell engager (BiTE®) antibody constructs directed to CDH19 and DLL3 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 553.

Targeted therapy that inhibits the BRAF (V600E) protein has shown impressive initial responses in metastatic melanoma patients. However, acquired drug resistance, derived from tumor heterogeneity, always limits its efficacy and compromises its treatment outcomes. Melanocyte to neural crest transition (MNT) has been observed to play crucial role in early stage adaptive drug tolerance, and the mathematic modeling indicates the transition is contributed from both stochastic phenotypic transition and non-genetic selection. Whole transcriptomic analysis enriched important signaling proteins that are involved in the resistance development process. Single cell proteomic analysis of those proteins was performed on patient-derived melanoma models to further study the early stage drug tolerance. This resolves how protein signaling network coordination rewires in the process, which cannot be resolved from proteomic analysis of bulk tumor tissue. Signaling network coordination analysis indicates that co-inhibiting the hubs of the network (MEK, NFKB-p65) could keep cells in the drug-susceptible stages. The combination therapy that co-inhibits mutant BRAF, MEK and NFKB-p65 together successfully arrested the MNT transition and induced a sustained tumor growth suppression in multiple patient-derived melanoma cell lines. This study provides us a brand-new way of using single cell proteomic analysis to infer effective combinations by resolving protein signaling network coordination that is inaccessible through traditional bulk-level proteomic measurement. Citation Format: Yapeng Su, Wei Wei, Min Xue, Lidia Robert, Jennifer Tsoi, Thomas Graeber, Antoni Ribas, James Heath. Single cell analysis resolves combinatory targeted therapy for arresting the BRAFi-induced cellular dedifferentiation of metastatic melanomas. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 879.

Melanoma can adapt to several stress factors, such as TNF treatment, by changing its phenotype from a highly differentiated (MITF and MART-1 positive) to undifferentiated (neural crest markers) states. We studied this phenomenon as a mechanism of adaptive resistance to BRAF inhibition since melanomas with both MITF high and low have been reported to result in BRAF inhibitor resistance. We treated 17 BRAFV600E mutant melanoma cell lines with the BRAF inhibitor vemurafenib for short term (3 days) or long term (17 days), and compared them to no treatment or TNF treated conditions. We profiled them phenotypically for MART1 antigen (differentiated phenotype) and nerve growth factor receptor (NGFR, neural crest phenotype) expression by flow cytometry. Results comparing median fluorescence intensity (MFI) showed a wide spectrum of adaptive responses to short term BRAF inhibition, followed by solidification or further evolution of these changes upon long term exposure and development of drug tolerance. According to each cell line distribution, we calculated a (x,y) value that organized the cell lines and conditions in four quadrants according to MART1 (x axis) or NGFR (y axis) expression. Two double negative (MART1-/NGFR-) cell lines that were unaffected by treatment and stayed in the same quadrant. Seven MART1-/NGFR+ and four MART1+/NGFR- cell lines slightly changed phenotype with vemurafenib exposure but stayed in the same quadrant. Four cell lines with MART1+/NGFR+ phenotype were greatly influenced by treatment, showing wide plasticity with cells shifting towards NGFR+/MART1- and others towards NGFR-/MART1+. Changes in position between baseline and tolerance to vemurafenib after 17days treatment were numerically quantified as an inferred number from plasticity. Cell lines with double positive (MART1+/NGFR+) phenotype had a significant change in plasticity compared to the rest of cell lines in other quadrants (p = 0.0004). This arranges cells in a four-quadrant pattern that could be likened to a U-turn toward de-differentiation. Cells that de-differentiated with BRAF inhibition mimicked the effect induced by TNF. This effect was driven by a decrease in MITF activity confirmed with a tyrosinase promoter-luciferase assay. In conclusion, baseline melanoma differentiation phenotypes guide the phenotype switch response, with those double positive for differentiation and neural crest markers being at a semi-stable state that can be prone to different plastic changes upon BRAF inhibition. Citation Format: Lidia Robert, Jennifer Tsoi, Angel Garica Diaz, Blanca Homet Moreno, Nhat Truong, Thomas Graeber, Antoni Ribas. Melanoma phenotype switching to adapt to BRAF inhibition. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2682. doi:10.1158/1538-7445.AM2015-2682

BRAF inhibitor targeted therapies such as vemurafenib (PLX4032) or dabrafenib, block BRAFV600 driver oncogenic mutations and result in high initial response rates and improve survival in patients with melanoma. Although response rate is over 50%, resistance eventually develops. A possible explanation is established tumors escape supported by the immune suppressive tumor-infiltrating myeloid cells (TIMs) including M2 type macrophages and myeloid derived suppressor cells (MDSC). Our murine melanoma SM1 model that harbors BRAFV600 mutation is an aggressive model, where mice with established tumors have to be sacrificed within two-three weeks. Treatment with PLX4032 has significant anti-tumor effect, but it cannot fully eradicate the tumor. In addition, our prior results show that SM1 is infiltrated by M2-polarized macrophages. By depleting TIMs with the CSF-1R inhibitor, PLX3397, the tumor infiltrating lymphocytes (TILs) is increased. To determine if combined therapy with PLX3397 and PLX4032 improved anti-tumor responses against SM1 tumors, C57BL/6 mice with established subcutaneous SM1 tumors received oral gavage daily with PLX3397 (50mg/kg) and i.p. daily with PLX4032 (100mg/kg). Combined treatment demonstrated superior effects compared to either of single treatment (tumor size on day 12_vehicle: 862.0±17.0 mm2, PLX3397: 439.3±6.9 mm2, PLX4032: 113.7±2.6 mm2, PLX3397+PLX4032: 12.9±4.6 mm2, p0.05). To further confirm that combined treatment effect is mediated by CD8+ T-cells, SM1 tumors were implanted in immunocompetent C57BL/6 mice and treated with PLX3397, PLX4032, and anti-CD8 neutralizing antibody. The anti-tumor response in PLX3397, PLX4032, and anti-CD8 antibody group was greatly diminished (tumor size on day 16_vehicle: 751.6±12.2 mm2, PLX4032: 232.4±8.8 mm2, anti-CD8: 722.8±12.5 mm2, PLX3397+PLX4032: 101.0±7.6 mm2; PLX3397+PLX4032+anti-CD8: 442.0±5.7 mm2, p>0.05). In conclusion, our data from SM1 in vivo model combining CSF-1R blockade and BRAF oncogenic blockade supports the rationale for testing this combination in patients with advanced melanoma, and suggest that the benefit is mediated by anti-tumor T cells. This combination is now in clinical testing (NCT01826448). Citation Format: Stephen Mok, Richard Koya, Jennifer Tsoi, Lidia Robert, Jesse Zaretsky, Christopher Tsui, Thomas Graeber, Antoni Ribas. Improving antitumor effects of a BRAF inhibitor with a colony stimulating factor 1 receptor (CSF-1R) inhibitor, PLX3397. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 147. doi:10.1158/1538-7445.AM2014-147

MAPK pathway dysregulation via mutations in proteins such as BRAFV600E and NRASQ61 is a key feature in melanomas, leading to constitutive ERK signaling. Currently, there are no FDA-approved targeted therapies effective in vemurafenib-resistant BRAFV600E mutant, NRAS mutant, or wild-type melanoma. The effect of SCH722984, an allosteric inhibitor of ERK 1/2, was determined in a panel of 53 melanoma cell lines, including wild-type, NRAS-mutant and vemurafenib-sensitive and resistant BRAF-mutant melanoma. 54% (13 of 24) of BRAF mutants, including 4 with innate vemurafenib resistance, were sensitive to SCH722984 with an IC50 Citation Format: Deborah J. Wong, Lidia Robert, Mohammad S. Atefi, David Foulad, Earl V. Avramis, Begonya Comin-Anduix, Ahmed A. Samatar, Antoni Ribas. High antitumor activity of the ERK inhibitor SCH722984 against BRAF-mutant, NRAS-mutant and wild-type melanoma cell lines. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 917. doi:10.1158/1538-7445.AM2013-917

The BRAF V600E driver mutation is found in more than 50% of melanomas. Less common BRAF mutations of V600K, V600R, L597S, and G466K have also been identified. In these rare BRAF mutants, the effects of vemurafenib (Vem) and other MAPK pathway inhibitors on signaling, growth rate and clinical course are not well understood. In cell lines with rare BRAF mutations, we investigated the effect of Vem, inhibitors of Pan-RAF (Amgen 2112819), MEK (trametinib), ERK (MERK SCH772984), Pan-RAFi+ MEKi and ERKi+Vem on growth and signaling. These results were compared to the clinical responses to BRAF and MEK inhibitors in patients with the original tumors. Regardless of the mutation, no paradoxical activation of MAPK pathway by Vem was observed and the Pan-Rafi decreased levels of pMEK in all the cell lines. A heterozygous V600K mutated cell line was sensitive to Vem and more sensitive to the other drugs. Clinically, the duration of response to Vem in the patient the cell line was derived from was 7 months, and an additional 3 months to the combination of Vem and MEKi upon progression on Vem. In contrast, a homozygous V600K mutated cell line was resistant to Vem but sensitive to other MAPK inhibitors. The patient the cell line was derived from had a 5 month response to Vem followed by a 2 month response to the addition of a MEKi. BRAF L597S cell line was only sensitive to ERKi and G466K and V600R mutated cell lines were resistant to all the drugs. In general, Vem was the least effective followed by Pan-RAFi with ERKi stronger than both in most cell lines. The combination of Vem and ERKi showed similar or slightly higher growth inhibition than ERKi alone. In conclusion, MAPK inhibitors are not as effective in melanoma cell lines with rare BRAF mutations. However, more growth inhibition can be achieved by MEKi-Pan-RAFi combination. These in vitro data seem to be correlated with the clinical outcomes. *Charles Ng, equal contribution Citation Format: Mohammad S. Atefi, Charles P. Ng, Deborah J. Wong, Lidia Robert, Helena Escuin-Ordinas, Amanda Lassen, David Foulad, Begonya Comin-Anduix, Ahmad A. Samatar, Antoni Ribas. Melanomas with rare BRAF mutations and their responses to MAPK pathway blocking drugs. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 915. doi:10.1158/1538-7445.AM2013-915