Oncogenes and Tumor Suppressors Biochemical and Structural Analysis of Common Cancer-Associated KRAS Mutations

Abstract KRAS mutations are the most common genetic abnormalities in cancer, but the distribution of specific mutations across cancers and the differential responses of patients with specific KRAS mutations in therapeutic clinical trials suggest that different KRAS mutations have unique biochemical behaviors. To further explain these high-level clinical differences and to explore potential therapeutic strategies for specific KRAS isoforms, we characterized the most common KRAS mutants biochemically for substrate binding kinetics, intrinsic and GTPase-activating protein (GAP)-stimulated GTPase activities, and interactions with the RAS effector, RAF kinase. Of note, KRAS G13D shows rapid nucleotide exchange kinetics compared with other mutants analyzed. This property can be explained by changes in the electrostatic charge distribution of the active site induced by the G13D mutation as shown by Xray crystallography. High-resolution X-ray structures are also provided for the GDP-bound forms of KRAS G12V, G12R, and Q61L and reveal additional insight. Overall, the structural data and measurements, obtained herein, indicate that measurable biochemical properties provide clues for identifying KRASdriven tumors that preferentially signal through RAF. Implications: Biochemical profiling and subclassification of KRAS-driven cancers will enable the rational selection of therapies targeting specific KRAS isoforms or specific RAS effectors

YB-1 transcription factor promotes Sorafenib resistance in Liver Cancer

Background: Hepatocellular carcinoma (HCC) is a primary malignant liver tumor that commonly occurs as a progression of chronic liver inflammation. Sorafenib is the standard first-line systemic drug for advanced HCC, but the acquired resistance to sorafenib results in limited benefits. The mechanism underlying sorafenib resistance in HCC remains unclear. Recently, we have identified a multifunctional oncoprotein Y-box binding protein-1 (YB-1) that dysregulates a wide range of genes involved in drug resistance in other cancers and is responsible for increasing the IC-50 of sorafenib in HCC cell lines. In this study we will analyze the signaling pathways and genes regulated by YB-1, that is responsible for increasing sorafenib resistant in liver cancer cells. Methods: HCC cell lines SK-Hep-1, C3A, HepG2 and Hep-3B were treated with Sorafenib and the IC-50 was calculated using MTT assay. RNA and protein of YB-1 was analyzed using RT-PCR and western blot respectively. Lentiviral based overexpression and knockdown of YB1 was performed in these cell lines and sorafenib IC50 were calculated to verify its role in Sorafenib resistance. Development of sorafenib resistant cell line is in progress. Results: IC-50 values calculated from MTT assays of the HCC cell lines were compared with the YB-1 protein expression in four liver cancer cell lines. Knockdown of YB-1 re-sensitized cell lines to Sorafenib. We have developed Sorafenib resistant cell lines to further study the mechanism of YB-1 mediated drug resistance. Conclusion: This study will establish oncogenic YB-1 protein as an effective therapeutic target to overcome sorafenib resistance in liver cancer

KRAS Switch Mutants D33E and A59G Crystallize in the State 1 Conformation

KRAS switch loop movements play a crucial role in regulating RAS signaling, and alteration of these sensitive dynamics is a principal mechanism through which disease-associated RAS mutations lead to aberrant RAS activation. Prior studies suggest that despite a high degree of sequence similarity, the switches in KRAS are more dynamic than those in HRAS. We determined X-ray crystal structures of the rare tumorigenic KRAS mutants KRAS^D33E, in switch 1 (SW1), and KRAS^A59G, in switch 2 (SW2), bound to GDP and found these adopt nearly identical, open SW1 conformations as well as altered SW2 conformations. KRAS^A59G bound to a GTP analogue crystallizes in the same conformation. This open conformation is consistent with the inactive “state 1” previously observed for HRAS bound to GTP. For KRAS^A59G, switch rearrangements may be regulated by increased flexibility in the ⁵⁷DXXGQ⁶¹ motif at codon 59. However, loss of interactions between side chains at codons 33 and 35 in the SW1 ³³DPT³⁵ motif drives changes for KRAS^D33E. The ³³DPT³⁵ motif is conserved for multiple members of the RAS subfamily but is not found in RAB, RHO, ARF, or Gα families, suggesting that dynamics mediated by this motif may be important for determining the selectivity of RAS–effector interactions. Biochemically, the consequence of altered switch dynamics is the same, showing impaired interaction with the guanine exchange factor SOS and loss of GAP-dependent GTPase activity. However, interactions with the RBD of RAF are preserved. Overall, these observations add to a body of evidence suggesting that HRAS and KRAS show meaningful differences in functionality stemming from differential protein dynamics independent of the hypervariable region

Suppressed rf dissipation in 107Ag17+^{107}Ag^{17+} ion irradiated Bi2Sr2CaCu2O8Bi_2Sr_2CaCu_2O_8 single crystals by enhanced flux line tilt modulus

We have studied the isothermal, magnetic field $(H\parallel c)$ dependent rf power P(H) dissipation $(H_{rf}\parallel a)$ in the superconducting state of $Bi_2Sr_2CaCu_2O_8$ single crystals prior to and after irradiation with 250 MeV $^{107}Ag^{17+}$ ions. In the pristine state, P(H) shows an initial decrease with increase in field, reaches a minimum at $H_M(T)$ and increases monotonically for H\lessthanH_M(T). This behavior arises when the electromagnetic coupling between the pancake vortices in adjacent CuO layers becomes dominant on increasing the field and minimizes the distortions of the flux lines by confining the 2D vortices. In the post irradiated state, such an initial decrease and the minimum in P(H) is not observed but only a much reduced rf dissipation that monotonically increases with field from H50 onwards is seen. We attribute this difference to the strong enhancement of the tilt modulus $C_{44}$ of the flux lines on irradiation when the pancake vortices in adjacent CuO bilayers are pinned along the track forming a well-stacked flux line in the field direction $(\parallel c)$. We have also observed that the rf dissipation disappears at a certain temperature $T_{sf}$ , at which the normal core of the flux line becomes commensurate with the columnar track diameter

Oxygen displacement in a 107Ag17+ ion irradiated Bi2Sr2CaCu2O8 single crystal

We have studied the magnetic field (H∥c) dependent rf dissipation (Hrf∥a) in an as-grown Bi2Sr2CaCu2O8 single crystal prior to and after irradiation with 250 MeV 107Ag17+ ions. In a comparison of the responses from the as-grown crystal with an air-annealed crystal, features due to oxygen deficient regions acting as weak links in the former are identified. These features disappear immediately after irradiation of the as-grown crystal. We attribute such behavior to the displacement of oxygen from columnar tracks to deficient regions thus eliminating the weak links. Losses from the same irradiated as-grown crystal stored at 300 K for 60 days show that the features similar but not identical to those observed in the pristine state have reappeared implying that the displaced oxygen is in a metastable configuration in the deficient regions and hence is mobile due to thermal effects even at 300 K