Macropinocytosis As a Means of Selectively Targeting RAS-Mutant Multiple Myeloma
Authors: Beals Nathan , Ramirez Craig , Hauser Andy , Davies Faith E. , Bar-Sagi Dafna , Morgan Gareth J. 
Published: December 2023

Introduction
Roughly, 50% of multiple myeloma (MM) is driven by mutant RAS, both at presentation and more at relapse. Currently, in MM there are no clinical approaches to target this disease subset and ongoing strategies to inhibit RAS function or downstream effectors have had limited clinical success in other tumors. This has prompted our team to look into other more indirect modes of RAS-selective targeting. Oncogenic RAS-driven cancers use a unique actin-driven endocytosis pathway, termed macropinocytosis (MP), to increase the nutrient supply and sustain proliferation. To create a strategy for targeting RAS mutant MM, we have investigated whether RAS-mutant MM cells undergo MP and whether this process can be exploited as a therapeutic vulnerability, by utilizing this pro-oncogenic biochemical mechanism to selectively deliver a toxin to the MM clone using a MP selective monobody-drug conjugate (MDC).
Methods
The MDC relies on a proprietary fibronectin-based scaffold (monobody) identified based on its physiochemical properties that facilitates MP selective internalization, and therefore targets RAS-mutant MM tumor cells, with a very favorable pharmacokinetic profile. MMAE was attached to the monobody with a cathepsin-cleavable linker and a maleimide group for conjugation to the C-terminal free cysteine on the monobody protein (MDC-MMAE). MDC-MMAE was pre-clinically evaluated both in-vitro and in-vivo, in human MM cell lines, patient cells and a doxycycline inducible mutant KRAS cell system.
Results
In-vitro cell line and patient cell studies demonstrated that RAS-mutant MM cells undergo MP whereas wild type (WT) cells do not. To understand the role of mutant RAS in driving uptake of the MDC, a doxycycline-inducible mutant KRAS V12 KMS11 system was established. The functionality of the inducibility of the cell system was verified using protein expression and membrane ruffling in the presence of doxycycline. Fluorescently-labeled monobody (MDC-Cy5.5) and TMR-Dextran underwent a high level of uptake in the presence of induced mutant RAS. In contrast, in conditions where MP was absent, no uptake of MDC-Cy5.5 and TMR-Dextran was seen. Furthermore, when KMS11 KRASV12 and WT KMS11 were treated with MDC-MMAE, a 2-fold increase in cell death was observed in the mutant RAS cell line compared to wild type RAS.
In-vivo studies showed that the pharmacological properties of the drug when conjugated to the monobody enabled faster serum clearance compared to antibody drug conjugates (ADCs) and nanomaterials, while maintaining cellular impermeability allowing enhanced drug uptake via MP. Importantly, in-vivo the MDC-MMAE technology had an improved toxicity profile compared to ADC-MMAE, increasing tolerable drug delivery by upwards of 15-fold. To test the anti-tumor effects in-vivo, the L363 (NRAS Q61), WT KMS11, and KMS11 KRASV12 cell lines were subcutaneously injected into MM murine models. After tumors were established, 0.5mg/kg MDC-MMAE was administered every 7 days. Mutant RAS L363 and KMS11 KRASV12 tumors treated with MDC-MMAE exhibited roughly a 6-fold decrease in tumor size after 21 days while WT KMS11 tumors showed no significant difference in tumor volume. MDC-MMAE at 5mg/kg showed complete tumor regression as a single agent in L363 tumors. Improved outcomes were seen with MDC-MMAE in combination with standard of care options at lower doses. Additionally, MDC-MMAE has displayed enhanced immunogenic cell death that has not been seen with other chemotherapeutic options, making it an ideal partner for immunotherapy combination.
Conclusion
Limited clinical options exist for the treatment of mutant RAS MM tumors. We show that by taking advantage of MP, a process upregulated in RAS-mutant cancers, MDC-MMAE specifically targets and inhibits MM growth and improves survival in murine models. It can also be combined with other MM standard of care drugs and with immunotherapy drugs to have a potentially important clinical effect. In collaboration with Tezcat Biosciences, the MDC-MMAE technology has achieved a phase II SBIR for advancement to clinical application.