1. Targeting Chaperone Dependence in Rhabdomyosarcoma
Amit J. Sabnis MD, Bivona Laboratory
UCSF Helen Diller Family Comprehensive Cancer Center
UCSF Benioff Children’s Hospital

2. Two Paradigms for Developing Cancer Therapies
Oncogene Dependence
Non-Oncogene Dependence
The Bivona lab is interested in developing novel therapeutics for cancer patients by studying both oncogene and non-oncogene dependencies. In oncogene dependence, a model well characterized by mutant EGFR in lung adenocarcinoma, a mutant oncogene simultaneously promotes strong growth and survival signals while inhibiting death signals. Inhibition of the oncogene, for instance by treating with a tyrosine kinase inhibitor, leads to cell death. This is a viable therapeutic strategy when oncogenes are defined and pharmacologically actionable in human cancers.
By contrast, the concept of non-oncogene dependence as described by Stephen Elledge refers to a number of cancer “stress phenotypes” including oxidative stress, proteotoxic stress, and DNA replicative stress that many cancers share in common. Normal cellular buffers protect cancers from the ill effects of these stresses, raising the possibility of a “synthetic lethal” interaction when that buffer is removed, leading to the death of transformed but not normal cells.
Luo J et al., Cell 2009

3. HSP70 Chaperones are Cellular Buffers Against Proteotoxic Stress
Hypothesis: HSP70 inhibition will engage stress responses in rhabdomyosarcoma cells and lead to cell death.
One candidate buffer for therapeutic development is the heat shock protein 70-kD or HSP70 family. These proteins assist in the degradation or, through interaction with the better-studied HSP90, refolding of misfolded peptides. HSP70 is upregulated in a number of malignancies, suggesting a broad therapeutic relevance.
We hypothesized, then, that while active HSP70s allow cancer cells to survive the oncogenic stresses of aneuploidy, oxidative stress, and transcriptional stress, inhibition of HSP70 activity would engage normal stress response mechanisms leading to cancer cell death.
Blais A et al., Genes & Development 2005

4. The HSP70 Inhibitor MAL3-101 Induces Apoptosis in Rhabdomyosarcoma (RMS) Cells
RMS13 Cells: 24 Hr Treatment
As a tool to test this hypothesis, we used a pharmacologic inhibitor of HSP70’s ATPase activity, a compound called MAL We evaluated the cellular consequences of MAL3-101 treatment in a number of patient-derived cancer cell lines. We found that rhabdomyosarcoma cells were the most sensitive to the drug’s effects; shown here in black are two fusion-positive cell lines and one fusion-negative cell line. As an example, a rhabdoid tumor cell line (A204 in green) and a lung adenocarcinoma cell line (H3122 in blue) were quite resistant to the drug’s effects, even at higher doses.
Biochemically, we found evidence of apoptosis in sensitive lines. Shown here is a WB of RMS13 cells after 24 hours of treatment with DMSO, actinomycin D, or MAL3-101, showing robust PARP cleavage.
In order to develop this agent further for clinical use, we sought to understand the specific mechanisms underlying sensitivity to MAL3-101.
Patient-derived rhabdomyosarcoma cell lines RMS13, Rh41, and Rh18 show micromolar sensitivity to MAL3-101.
MAL3-101 induces apoptosis in sensitive cell lines.

5. The Integrated Stress Response (ISR) Links Chaperone Failure to Apoptosis
A possible mechanistic link between HSP70 inhibition and apoptosis lies in the integrated stress response pathway. This pathway can be activated by multiple stressors, including protein misfolding. Upon accumulation of misfolded peptides in the ER, the kinase PERK is activated and leads to phosphorylation of the translation initiation factor EIF2a. This in turn leads to global stalling of protein synthesis, but an increase in production of two key transcription factors. ATF4 is an early marker of ISR activation, and is thought to exert a primarily homeostatic role. By contrast, the transcription factor, best known to this audience as a fusion partner in liposarcoma, is in fact pro-apoptotic in the context of prolonged ISR signaling.
We hypothesized, then, that activation of the ISR is the cause of cell death after MAL3-101 treatment.
Hypothesis: Activation of the integrated stress response leads to cell death after MAL3-101 treatment.

6. Isogenic Resistant Lines
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
RMS13
I.R. #1 - 4
One validated approach to understanding response to a drug is to identify the mechanisms that promote resistance to it. Using a method widely used in the study of solid tumor cancer cell lines, we grew sensitive cells in escalating doses of MAL3-101 over two months. At the end of this experiment, we had four independently--derived isogenic resistant lines (I.R. #1-4) that we could treat with MAL3-101, and then compare cellular and biochemical responses to those of the parental line. This allows us to not only understand the mechanisms underlying drug sensitivity, but also to begin to model how drug resistance may emerge in the clinic.
Treat with MAL3-101
Measure Integrated Stress Response

7. ISR Activation is a Hallmark of MAL3-101 Sensitivity
We found that degree of ISR activation did indeed correlate with drug sensitivity. Here, a Western blot of sensitive RMS13 cells or isogenic resistant lines 1, 2, and 3 demonstrates PARP cleave in the parental line only. Similarly, eIF2a phosphorylation, a proximal readout of ISR activation, is significantly greater in the parental lines. We did find that levels of ATF4 did not differ significantly amongst parental and resistant lines, consistent with its role as an early and quite sensitive readout of ISR activation.

8. ISR Activation is a Hallmark of MAL3-101 Sensitivity
By contrast, quantitative PCR for the pro-apoptotic transcription factor CHOP demonstrated a clear difference between sensitive and resistant lines. Here, levels of CHOP transcript normalized to GAPDH are shown in 3-hour increments following MAL3-101 treatment in parental RMS13 or isogenic resistant lines. As you can see, IR #2 and #3 do not ever induce CHOP to the same extent as parental lines, whereas IR #1 has initial CHOP induction, but then attenuation of this signal within 12 hours.

9. Loss of CHOP Promotes Resistance to MAL3-101
CHOP Knockdown
by shRNA
Sensitivity to MAL3-101
We then asked whether CHOP was required for drug sensitivity by knocking this gene down by shRNA. On the left you can see levels of CHOP transcript by qPCR in parental cells in gray or knockdown cells in blue, confirming lower basal levels of CHOP as well as blunting of MAL3-101 induced upregulation. Viability curves of RMS13 cells, CHOP KD cells, and isogenic resistant lines after 72 hours of treatment show that CHOP knockdown is sufficient for promoting resistance to MAL3-101, though not to the same extent as in the derived lines. This suggests that additional mechanisms may also support the survival of these cells.

10. 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.

11. 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.
(e.g. IRE1 and ATF6 for unfolded protein response, NFE2L2 for oxidative stress, GCN2 for nutrient deprivation)

12. Acknowledgements Bivona Lab Support Collaborators Trever Bivona
Jonathan Shue
Anin Sayana
Luping Lin
Collin Blakely
Evangelos Pazarentzos
Support
HCIA Award (T.B.)
Damon Runyon-Sohn
Pediatric Cancer Fellow (A.J.S.)
Collaborators
Jeff Brodsky (U. Pittsburgh)
Chris Guerriero
Peter Walter (UCSF)
Diego Acosta-Alvear
Carmela Sidrauski
Jonathan Weissman (UCSF)
Jason Gestwicki (UCSF)
St. Baldrick’s
Foundation Fellow (A.J.S.)