Targeting Chaperone Dependence in Rhabdomyosarcoma

Amit J. Sabnis MD, Bivona Laboratory

UCSF Helen Diller Family Comprehensive Cancer Center

UCSF Benioff Children’s Hospital

Two Paradigms for Developing Cancer Therapies

Oncogene Dependence Non-Oncogene Dependence

Luo J et al., Cell 2009

HSP70 Chaperones are Cellular Buffers Against Proteotoxic Stress

Hypothesis: HSP70 inhibition will engage stress responses in rhabdomyosarcoma cells

and lead to cell death.

The HSP70 Inhibitor MAL3-101 Induces Apoptosis in Rhabdomyosarcoma (RMS) Cells

• Patient-derived rhabdomyosarcoma cell lines RMS13, Rh41, and Rh18 show micromolar sensitivity to MAL3-101.

• MAL3-101 induces apoptosis in sensitive cell lines.

RMS13 Cells: 24 Hr Treatment

The Integrated Stress Response (ISR) Links Chaperone Failure to Apoptosis

Hypothesis: Activation of the integrated stress response leads to cell death after MAL3-101 treatment.

Probing the Mechanism of Action of MAL3-101 with Derived Isogenic Resistant Cell Lines

Parental Line(MAL3-101 Sensitive)

Isogenic Resistant Lines(MAL3-101 Resistant)

2 Month MAL3-101

Dose Escalation

Treat with MAL3-101Measure Integrated Stress Response

RMS13 I.R. #1 - 4

ISR Activation is a Hallmark of MAL3-101 Sensitivity

ISR Activation is a Hallmark of MAL3-101 Sensitivity

Loss of CHOP Promotes Resistance to MAL3-101

CHOP Knockdown by shRNA Sensitivity to MAL3-101

Conclusions

• MAL3-101 is an HSP70 inhibitor that induces apoptosis in rhabdomyosarcoma cells.

• Apoptosis is a consequence of engaging the integrated stress response, and is CHOP dependent.

• Failure to upregulate CHOP after HSP70 inhibition is a biomarker of resistance to MAL3-101.

• Loss of CHOP causes partial resistance to MAL3-101.

Future Directions

• Test whether other stress sensors cooperate with the ISR in inducing apoptosis after HSP70 inhibition.

• Carry out an unbiased shRNA screen to comprehensively identify mechanisms underlying resistance in I.R. lines.

• Test MAL3-101 for activity in explant models of RMS as a prelude to clinical use.

Acknowledgements

Bivona LabTrever BivonaJonathan ShueAnin Sayana

Luping LinCollin BlakelyEvangelos Pazarentzos

Support

Damon Runyon-Sohn Pediatric Cancer Fellow (A.J.S.)

St. Baldrick’s Foundation Fellow (A.J.S.)

HCIA Award (T.B.)

CollaboratorsJeff Brodsky (U. Pittsburgh)

Chris Guerriero

Peter Walter (UCSF)Diego Acosta-AlvearCarmela Sidrauski

Jonathan Weissman (UCSF)

Jason Gestwicki (UCSF)