Catherine Burkhart1,2, Geraldine Paszkiewicz3, Loretta Gawron2, Rachael Kohrn2, Andrei Purmal1,4, Elizabeth Repasky3, Carl Morrison3, Angela Omilian3, Andrei Gudkov1,3,4 and Katerina Gurova3

1Cleveland BioLabs, Inc (Buffalo, NY, USA), 2Buffalo BioLabs, LLC (Buffalo, NY, USA), 3Roswell Park Cancer Institute (Buffalo, NY, USA), 4Incuron, LLC (Buffalo, NY, USA)

Background: CBL0137 represents a novel class of small molecules, Curaxins, that

simultaneously activate p53 and inhibit cancer-associated stress response

pathways, such as NF- B and HSF-11. CBL0137 antitumor activity has been

established in a broad range of tumor models both by oral and intravenous

administration1. The effects of CBL0137, culminating in tumor cell death, are

mediated by the sequestering of the FACT (FAcilitates Chromatin Transcription)

complex on chromatin thereby inhibiting its activity1. FACT is a transcription and

replication factor (composed of two subunits, Structure Specific Recognition

Protein (SSRP1) and suppressor of Ty 16 (SPT16)), which is involved in

transcription of genes with highly ordered chromatin structure as well as in

replication and mitosis1-3. FACT is expressed during early embryogenesis and in

undifferentiated progenitors and stem cells of adult tissues while protein levels

of both FACT subunits are almost undetectable in differentiated cells and

tissues2. FACT is expressed in several tumor types compared to equivalent

normal tissues. In addition, FACT positive tumors have an aggressive malignant

phenotype as illustrated by a correlation of FACT expression with high grade,

the development of metastatic disease and worse overall survival4. Thus, FACT

represents a potentially important target for cancer therapy. In particular, a high

proportion of pancreatic cancers express FACT as demonstrated by ~60% of

tested pancreatic tumors staining positive for the SSRP1 subunit (n=56)4.

Furthermore, 30% of these cases expressed a very high level of SSRP1.

1Gasparian et al (2011) Science Translational Medicine 3:95ra74 2Garcia et al (2011) Oncotarget 2:783-96 3Reinberg and Sims (2006) Journal Biological Chemistry 281:23297-23301 4Garcia et al (2013) Cell Reports (in press) http://dx.doi.org/10.1016/j.celrep.2013.06.013

METHODS: • Patient-derived pancreatic ductal adenocarcinoma (PDA) xenografts

were obtained under an approved IRB protocol and continually propagated in

SCID mice for use in efficacy experiments.

• Immunohistochemistry: TMAs containing 56 distinct PDA samples were

stained for SSRP1 and samples were scored for both % of positive cells and

intensity by trained a histopathologist.

• Pilot CBL0137 efficacy studies: SCID mice (n=5/treatment group; 2

tumors/mouse) were implanted with 2-5 mm3 of the designated patient PDA. ~10

days following implantation (tumor volumes <200 mm3) treatment commenced.

For oral administration, mice received vehicle or 30 mg/kg CBL0137 by gavage

following a 5 days on/2 days off schedule for 4 weeks. For iv studies, mice

received either vehicle or 90 mg/kg CBL0137 via the tail vein once per week up to

4 weeks. Tumors were measured 2-3 times per week using digital calipers. Tumor

volume was calculated using standard equation mm3 = L x W2/2 where L=

longest dimension and W is measured perpendicular to L. Mice were followed

until total tumor burden of mouse reached ~ 2000 mm3, all controls completed the

study or 29 days, whichever came first. Data is presented a mean fold tumor

growth by normalizing the tumor volume on Day X by that of Day 1. Comparisons

between treatment groups were performed by ANOVA.

• Combination efficacy studies: These studies were performed as described

above with the following modifications. Treatment commenced when the mean

tumor volume per group was ~25-50 mm3. Mice were treated with 80-90 mg/kg

CBL0137 iv (1/wk) in the presence or absence of 40 mg/kg gemcitabine

administered ip every 4th day (Q4d) for 4 weeks. Mice were followed until at least

one tumor per mouse reached 1000 mm3 or up to 90 days from start of treatment.

Normal CBL0137

NF B

p53

RNA - pol II

RNA

NF B RNA - pol II

p53 p53

p53 p53

p53 p53

p53 p53

p53 p53

p53 p53

RNA - pol II

nucleosome

CBL0137

Figure 1. Mechanism of action of CBL01371

Normal

PDA#13590 PDA#13756 PDA#15010

PDA#10978

Figure 3. SSRP1 Expression levels in patient derived PDA Xenografts

Figure 4. Effect of 30 mg/kg CBL0137 administered po (5 days on/2 days off for 4 weeks) on patient-derived PDA xenograft. Data is presented relative to Day 1 tumor volumes. * P<0.05, ANOVA.

PDA#12914

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* * * *

Oral Administration of CBL0137

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IV Administration of CBL0137

Figure 5. Effect of 90 mg/kg CBL0137 administered iv (once per week for up to 4 weeks) on 4 independent patient-derived PDA xenografts that were propagated through mice upon collection from patients. Studies for individual tumors ended when all control tumors reached the designated tumor volume endpoint. Data is presented relative to Day 1 tumor volumes. * P<0.05, ANOVA. N=10 tumors per treatment group.

Conclusions: • FACT represents a potential target for cancer therapy due to its level of expression compared to normal tissue,

association with an aggressive malignant phenotype, metastatic disease and poor overall survival (Garcia et al

(2013) Cell Reports (in press) http://dx.doi.org/10.1016/j.celrep.2013.06.013). It is found in a large proportion of

PDA.

• CBL0137, which targets FACT, caused a 49-76% reduction in FACT positive PDA xenografts derived from

patient tumors when administered orally daily or intravenously once per week.

• CBL0137 produced a similar antitumor effect to gemcitabine and Abraxane against a gemcitabine/Abraxane-

sensitive PDAs during the treatment period and appeared to enhance the antitumor effect of Abraxane and

delay the recurrence of tumors in combination with gemcitabine at a dose, in which it was ineffective itself.

Acknowledgments: This work was funded in part by an STTR grant awarded by the NCI (Buffalo BioLabs, LLC,

KG and CB) and a grant from Incuron, LLC (KG).

Sub-optimal CBL0137 dose significantly delays recurrence of gemcitabine-regressed patient-derived PDA xenografts

0 10 20 30 40 50 60 70 80 90 0

10 20 30 40 50 60 70 80 90

100 vehicle gemcitabine only CBL0137 only GEM + CBL0137

days from start of treatment

Perc

en

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PDA#13756

P=0.0153

Figure 7. Effect of 80 mg/kg CBL0137 on survival time of PDA-bearing mice in the presence or absence of gemcitabine.

Vehicle80 mg/kg CBL0137,

iv, 1/wk40 mg/kg

gemcitabine, ip, Q4dCBL0137 +

gemcitabine

Median survival time (days)

16 17 54 78

IV Administration of CBL0137 +/- Gemcitabine or +/- Abraxane

Figure 6. Effect of 80-90 mg/kg CBL0137 administered iv (1/wk for 4 weeks) ± 40 mg/kg gemcitabine (GEM) ip (Q4d for 4 weeks) or ± 30 mg/kg Abraxane (Abx) iv (D1-5) on patient derived PDA tumor growth.

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SSRP1 Expression levels in PDA Tissue Microarray Samples

SSRP1 Negative (<1)

SSRP1 + (>1)

High SSRP1

(>4)

Figure 2. TMAs including 56 PDA samples were stained for SSRP1 and scored based on % positive cells and intensity4.

BBL

Buffalo BioLabs, LLC