KRASG12D inhibition reprograms the microenvironment of early and advanced pancreatic cancer to promote FAS-mediated killing by CD8+ T cells.

The KRASG12D mutation is present in nearly half of pancreatic adenocarcinomas (PDAC). We investigated the effects of inhibiting the KRASG12D mutant protein with MRTX1133, a non-covalent small molecule inhibitor of KRASG12D, on early and advanced PDAC and its influence on the tumor microenvironment. Employing 16 different models of KRASG12D-driven PDAC, we demonstrate that MRTX1133 reverses early PDAC growth, increases intratumoral CD8+ effector T cells, decreases myeloid infiltration, and reprograms cancer-associated fibroblasts. MRTX1133 leads to regression of both established PanINs and advanced PDAC. Regression of advanced PDAC requires CD8+ T cells and immune checkpoint blockade (ICB) synergizes with MRTX1133 to eradicate PDAC and prolong overall survival. Mechanistically, inhibition of KRASG12D in advanced PDAC and human patient derived organoids induces FAS expression in cancer cells and facilitates CD8+ T cell-mediated death. Collectively, this study provides a rationale for a synergistic combination of MRTX1133 with ICB in clinical trials.

Elimination of oncogenic KRAS in genetic mouse models eradicates pancreatic cancer by inducing FAS-dependent apoptosis by CD8+ T cells.

Oncogenic KRASG12D (KRAS∗) is critical for the initiation and maintenance of pancreatic ductal adenocarcinoma (PDAC) and is a known repressor of tumor immunity. Conditional elimination of KRAS∗ in genetic mouse models of PDAC leads to the reactivation of FAS, CD8+ T cell-mediated apoptosis, and complete eradication of tumors. KRAS∗ elimination recruits activated CD4+ and CD8+ T cells and promotes the activation of antigen-presenting cells. Mechanistically, KRAS∗-mediated immune evasion involves the epigenetic regulation of Fas death receptor in cancer cells, via methylation of its promoter region. Furthermore, analysis of human RNA sequencing identifies that high KRAS expression in PDAC tumors shows a lower proportion of CD8+ T cells and demonstrates shorter survival compared with tumors with low KRAS expression. This study highlights the role of CD8+ T cells in the eradication of PDAC following KRAS∗ elimination and provides a rationale for the combination of KRAS∗ targeting with immunotherapy to control PDAC.

MMP modulated differentiation of mouse embryonic stem cells on engineered cell derived matrices.

Stem cell differentiation is dictated by the dynamic crosstalk between cells and their underlying extracellular matrix. While the importance of matrix degradation mediated by enzymes such as matrix metalloproteinases (MMPs) in the context of cancer invasion is well established, the role of MMPs in stem cell differentiation remains relatively unexplored. Here we address this question by assaying MMP expression and activity during differentiation of mouse embryonic stem cells (mESCs) on mouse embryonic fibroblast (MEF) derived matrices (MEFDMs) of varying stiffness and composition. We show that mESC differentiation into different germ layers is associated with expression of several MMPs including MMP-11, 2, 17, 25 and 9, with MMP-9 detected in cell secreted media. Different extents of softening of the different MEFDMs led to altered integrin expression, activated distinct mechanotransduction and metabolic pathways, and induced expression of germ layer-specific markers. Inhibition of MMP proteolytic activity by the broad spectrum MMP inhibitor GM6001 led to alterations in germ layer commitment of the differentiating mESCs. Together, our results illustrate the effect of MMPs in regulating mESC differentiation on engineered cell derived matrices and establish MEFDMs as suitable substrates for understanding molecular mechanisms regulating stem cell development and for regenerative medicine applications.

Fmn2 Regulates Growth Cone Motility by Mediating a Molecular Clutch to Generate Traction Forces.

Growth cone-mediated axonal outgrowth and accurate synaptic targeting are central to brain morphogenesis. Translocation of the growth cone necessitates mechanochemical regulation of cell-extracellular matrix interactions and the generation of propulsive traction forces onto the growth environment. However, the molecular mechanisms subserving force generation by growth cones remain poorly characterized. The formin family member, Fmn2, has been identified earlier as a regulator of growth cone motility. Here, we explore the mechanisms underlying Fmn2 function in the growth cone. Evaluation of multiple components of the adhesion complexes suggests that Fmn2 regulates point contact stability. Analysis of F-actin retrograde flow reveals that Fmn2 functions as a clutch molecule and mediates the coupling of the actin cytoskeleton to the growth substrate, via point contact adhesion complexes. Using traction force microscopy, we show that the Fmn2-mediated clutch function is necessary for the generation of traction stresses by neurons. Our findings suggest that Fmn2, a protein associated with neurodevelopmental and neurodegenerative disorders, is a key regulator of a molecular clutch activity and consequently motility of neuronal growth cones.

Initial Priming on Soft Substrates Enhances Subsequent Topography-Induced Neuronal Differentiation in ESCs but Not in MSCs.

Differentiation of stem cells into neurogenic lineage is of great interest for treatment of neurodegenerative diseases. While the role of chemical cues in regulating stem cell fate is well appreciated, the identification of physical cues has revolutionized the field of tissue engineering leading to development of scaffolds encoding one or more physical cues for inducing stem cell differentiation. In this study, using human mesenchymal stem cells (hMSCs) and mouse embryonic stem cells (mESCs), we have tested if stiffness and topography can be collectively tuned for inducing neuronal differentiation by culturing these cells on polyacrylamide hydrogels of varying stiffness (5, 10, and 20 kPa) containing rectangular grooves (10, 15, and 25 µm in width). While hMSCs maximally elongate and express neuronal markers on soft 5 kPa gels containing 10/15 µm grooves, single mESCs are unable to sense topographical features when cultured directly on grooved gels. However, this inability to sense topography is rescued by priming mESCs initially on soft 1 kPa flat gels and then replating these cells onto the grooved gels. Compared to direct culture on the grooved gels, this sequential adaptation increases both viability as well as neuronal differentiation. However, this two-step process does not enhance neuronal marker expression in hMSCs. In addition to highlighting important differences between hMSCs and mESCs in their responsiveness to physical cues, our study suggests that conditioning on soft substrates is essential for inducing topography-mediated neuronal differentiation in mESCs.

Deciphering the mechanism of potent peptidomimetic inhibitors targeting plasmepsins - biochemical and structural insights.

Malaria is a deadly disease killing worldwide hundreds of thousands people each year and the responsible parasite has acquired resistance to the available drug combinations. The four vacuolar plasmepsins (PMs) in Plasmodium falciparum involved in hemoglobin (Hb) catabolism represent promising targets to combat drug resistance. High antimalarial activities can be achieved by developing a single drug that would simultaneously target all the vacuolar PMs. We have demonstrated for the first time the use of soluble recombinant plasmepsin II (PMII) for structure-guided drug discovery with KNI inhibitors. Compounds used in this study (KNI-10742, 10743, 10395, 10333, and 10343) exhibit nanomolar inhibition against PMII and are also effective in blocking the activities of PMI and PMIV with the low nanomolar Ki values. The high-resolution crystal structures of PMII-KNI inhibitor complexes reveal interesting features modulating their differential potency. Important individual characteristics of the inhibitors and their importance for potency have been established. The alkylamino analog, KNI-10743, shows intrinsic flexibility at the P2 position that potentiates its interactions with Asp132, Leu133, and Ser134. The phenylacetyl tripeptides, KNI-10333 and KNI-10343, accommodate different ρ-substituents at the P3 phenylacetyl ring that determine the orientation of the ring, thus creating novel hydrogen-bonding contacts. KNI-10743 and KNI-10333 possess significant antimalarial activity, block Hb degradation inside the food vacuole, and show no cytotoxicity on human cells; thus, they can be considered as promising candidates for further optimization. Based on our structural data, novel KNI derivatives with improved antimalarial activity could be designed for potential clinical use. DATABASE: Structural data are available in the PDB under the accession numbers 5YIE, 5YIB, 5YID, 5YIC, and 5YIA.

Biophysical regulation of mouse embryonic stem cell fate and genomic integrity by feeder derived matrices.

For maintaining pluripotency, mouse embryonic stem cells (mESCs) are typically grown on mitotically inactivated mouse embryonic fibroblasts (MEFs). While the role of MEF conditioned media (MEFCM) and leukemia inhibitory factor (LIF) in regulating mESC pluripotency has led to culturing of mESCs on LIF/MEFCM supplemented gelatin-coated substrates, the role of physical interactions between MEFs and mESCs in regulating mESC pluripotency remains to be fully understood. Here, we address this question by characterizing the physicochemical properties of MEF derived matrices (MEFDMs), and probing their role in regulating mESC fate. We show that MEFDM composition and stiffness-dictated by MEF contractility-regulates mESC pluripotency by modulating mESC contractility through integrin-mediated mechanoadaptation. While baseline mESC pluripotency is maintained at early time points, activation of mESC contractility by LPA leads to drop in pluripotency levels. In contrast, addition of blebbistatin and LIF independently increases pluripotency by suppressing mechanoadaptation, highlighting the role of mechanoadaptation in regulating pluripotency and illustrating the role of LIF as a mechano-inhibitor in mESCs. Long-term culture of mESCs on MEFDMs under LIF-free conditions triggers loss of pluripotency, and induces ligand-dependent expression of the osteogenic transcription factor Runx2. Maintenance of genomic integrity (euploidy) on MEFDMs but not on gelatin-coated substrates, combined with the ability of MEFDMs in supporting LIF-free expansion and differentiation of mESCs, illustrates the suitability of MEFDMs for clinical and regenerative medicine applications.

Membrane Vesicles of Group B Streptococcus Disrupt Feto-Maternal Barrier Leading to Preterm Birth.

Infection of the genitourinary tract with Group B Streptococcus (GBS), an opportunistic gram positive pathogen, is associated with premature rupture of amniotic membrane and preterm birth. In this work, we demonstrate that GBS produces membrane vesicles (MVs) in a serotype independent manner. These MVs are loaded with virulence factors including extracellular matrix degrading proteases and pore forming toxins. Mice chorio-decidual membranes challenged with MVs ex vivo resulted in extensive collagen degradation leading to loss of stiffness and mechanical weakening. MVs when instilled vaginally are capable of anterograde transport in mouse reproductive tract. Intra-amniotic injections of GBS MVs in mice led to upregulation of pro-inflammatory cytokines and inflammation mimicking features of chorio-amnionitis; it also led to apoptosis in the chorio-decidual tissue. Instillation of MVs in the amniotic sac also resulted in intrauterine fetal death and preterm delivery. Our findings suggest that GBS MVs can independently orchestrate events at the feto-maternal interface causing chorio-amnionitis and membrane damage leading to preterm birth or fetal death.

Probing cellular mechanoadaptation using cell-substrate de-adhesion dynamics: experiments and model.

Physical properties of the extracellular matrix (ECM) are known to regulate cellular processes ranging from spreading to differentiation, with alterations in cell phenotype closely associated with changes in physical properties of cells themselves. When plated on substrates of varying stiffness, fibroblasts have been shown to exhibit stiffness matching property, wherein cell cortical stiffness increases in proportion to substrate stiffness up to 5 kPa, and subsequently saturates. Similar mechanoadaptation responses have also been observed in other cell types. Trypsin de-adhesion represents a simple experimental framework for probing the contractile mechanics of adherent cells, with de-adhesion timescales shown to scale inversely with cortical stiffness values. In this study, we combine experiments and computation in deciphering the influence of substrate properties in regulating de-adhesion dynamics of adherent cells. We first show that NIH 3T3 fibroblasts cultured on collagen-coated polyacrylamide hydrogels de-adhere faster on stiffer substrates. Using a simple computational model, we qualitatively show how substrate stiffness and cell-substrate bond breakage rate collectively influence de-adhesion timescales, and also obtain analytical expressions of de-adhesion timescales in certain regimes of the parameter space. Finally, by comparing stiffness-dependent experimental and computational de-adhesion responses, we show that faster de-adhesion on stiffer substrates arises due to force-dependent breakage of cell-matrix adhesions. In addition to illustrating the utility of employing trypsin de-adhesion as a biophysical tool for probing mechanoadaptation, our computational results highlight the collective interplay of substrate properties and bond breakage rate in setting de-adhesion timescales.