Amaravadi Lab

Amaravadi Lab

The Amaravadi Lab focuses on developing new cancer therapy that addresses therapy resistance. Cancer therapy has made major strides in the past 20 years, but eventually most cancers have the ability to survive the stress of cancer therapy and recur, leading to suffering and death. We and others have demonstrated that autophagy, the process by which organelles such as mitochondria and proteins are internally digested and recycled, promotes cancer cell survival in advanced cancers, and is a potentially key resistance mechanism that cancers use to survive cancer therapy.  Our lab collaborates closely with other basic scientists, clinical researchers, biotechnology and pharmaceutical companies so that we can test our hypotheses in cell lines, 3D culture, mouse models, and patients. We conduct basic laboratory research to understand fundamental aspects of autophagy in cancer biology, but we also have a translational focus. We are actively conducting innovative clinical trials intended to modulate autophagy to enhance effectiveness of existing therapies. Our funding comes largely from the National Cancer Institute, focused pharmaceutical collaborations, and private donations from patients and fundraising programs. 

Past Projects

Can more potent and specific lysosomal autophagy inhibitors be identified?

Working with the laboratory of Dr. Jeffrey Winkler (Merriam Professor of Organic Chemistry Department of Chemistry, School of the Arts and Sciences University of Pennsylvania) we have designed, synthesized and tested the first series of dimeric chloroquine derivatives. We call these compounds Lys01 derivatives (see Mcafee et al PNAS 2012). We have identified dozens of potent derivatives (Rebecca et al. Cancer Discovery 2017; Rebecca et al. Cancer Discovery 2019) and identified the molecular target of chloroquine derivatives as palmitoyl-protein thioesterase 1 (PPT1). PPT1 inhibitors are being developed for clinical trials by Pinpoint Therapeutics, Inc.

What are the effects of lysosomal autophagy inhibition in specific cell types within the tumor microenvironment?

We recently showed that PPT1 inhibition in tumor associated macrophages enables T cell mediated killing of melanoma tumors (Sharma et al. JCI Insight 2020). Numerous groups have demonstrated that autophagy inhibition at other nodes in the pathway augments anti-tumor immunity. More work is needed to understand how best to use autophagy inhibitors in the context of immunotherapy.

What is the role of PPT1 in tumorigenesis?

Using CRISPR technology we have created novel tools to study the role of PPT1 in tumorigenesis .  CRISPR/Cas9 technology was used to insert LoxP sites around exon 1 of the mouse Ppt1 gene to generate conditional Ppt1 knockout mice.  Then the mice were crossed into a tamoxifen inducible model of melanoma tumorigenesis.  In vivo loss of PPT1 had no effect on initiation or growth of these tumors. CRISPR/Cas9 was also used to knockdown expression of PPT1 in human and mouse melanoma cell lines both constitutively and acutely (Dox inducible).  While chemical inhibition of PPT1 with DC661 is toxic to melanoma cells, genetic inhibition was not. Comparison of cells with knockdown by CRISPR/Cas9, siRNA or chemical inhibition indicated distinct proteome changes to adapt to loss of PPT1 which has implications for therapeutic use of PPT1 inhibitors. (Crissey, et. al.  Autophagy 2025)

Can we find the cancers and patients most likely to benefit from lysosomal autophagy inhibition?

Using -omics platforms we are investigating, genetic and epigenetic determinants of sensitivity to chloroquine derivatives in a broad panel of tumor cell lines and patient tissues. This work is in collaboration with investigators from the  Abramson Family Cancer Research Institute and the Center for Clinical Epidemiology and Biostatistics. Preliminary findings were published (Piao Autophagy 2017), but we continue to investigate candidate markers of sensitivity in human tumor samples. 

Can proteins that are secreted in an autophagy dependent manner be used to develop biomarkers of response to autophagy inhibitors? 

Working with laboratory of Dr. David Speicher (Caspar Wistar Professor of Computational and Systems Biology, Wistar Institute), we have probed the secretome of melanoma cells grown in 3D culture and have identified autophagy dependent secreted proteins (Kraya et al Autophagy 2015). We are currently determining effects of therapeutics that are known to modulate autophagy on secretome profiles. We are working on both small molecule targeted therapeutics and immunotherapies in immunocompetent mouse models.  

How exactly does targeted therapy activate a cytoprotective autophagy response?