Work in Progress

Hafsa Shafique
20 02 2023
 Outgoing plans
 CPSF73 is helpful in polyadenylation of nascent RNA.
 UBE3D, participates in stabilizing CPFS73 protein by preventing its
ubiquitin-mediated degradation by the proteasome.
 Depletion of UBE3D leads to CPSF73 downregulation.
 our findings indicate that targeting the pre-mRNA processing
nuclease CPSF73 has potential for breast cancer therapy.
cleavage and polyadenylation specificity factor (CPSF)
 We generated a HEK293 UBE3D knockout (KO) cell line using
CRISPR-Cas9 technology..
 UBE3D depletion in the KO cell line was verified both at the
genomic level and protein level.
UBE3D positively regulates CPSF73 but not other C/P proteins at the post-translational level

 UBE3D depletion does not affect transcription or stability of CPSF73 mRNAs.

UBE3D KO leads to impaired 3′ end processing, transcriptional readthrough of PASs, and
dysregulated gene expression

 Cycloheximide (CHX) chase assay revealed that CPSF73 is less stable in UBE3D KO cells.
 Glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
UBE3D depletion inhibits breast cancer cell motility in a CPFS73-dependent manner

• The level of CPFS73 in UBE3D KO cells was increased by the proteasome inhibitor MG132 and the E1 ubiquitin activating
enzyme inhibitor TAK243
Catalytic inhibition of CPSF73 leads to breast cancer cell migration and invasion defects
CPSF73 expression level correlates with breast cancer cell self-renewal properties
Further work

targeting CPSF73 in vivo to inhibit breast cancer

growth.
Research Plan on breast
cancer by targeting CPSF73
The mRNA endonuclease CPSF73 is an enzyme of the pre-mRNA 3' end
processing machinery and is known to be overexpressed in several types of
cancer, including breast cancer. Inhibition of CPSF73 activity has been
shown to decrease the proliferation of breast cancer cells.

These studies aim to assess the safety, efficacy, and pharmacokinetics of CPSF73
inhibitors, as well as their potential to overcome resistance to other breast cancer
therapies.
Mouse Model

an orthotopic mouse model
studying the targeting of CPSF73 in breast cancer.
can be a suitable choice for

 In an orthotopic model, human breast cancer cells are injected into the mammary gland of mice, mimicking the natural
growth and spread of breast cancer in humans.

 This allows for the study of the targeting of CPSF73 in a context that more closely resembles human breast cancer.

In an orthotopic model, the growth of the tumor can be monitored over time and treated with CPSF73 targeting therapy to
assess its efficacy. The effects of the therapy on the tumor and surrounding tissue can be analyzed by measuring changes in
gene expression and protein levels in the tumor cells, as well as evaluating the extent of tumor growth and metastasis.

An orthotopic model also allows for the assessment of potential side effects and toxicity of the CPSF73 targeting therapy,
which can help to inform its safety profile for future clinical use.
Overall, an orthotopic mouse model can provide valuable insights into the efficacy and safety of CPSF73 targeting therapy for
breast cancer, making it a useful tool for preclinical research
CPSF73 targeting therapy
siRNA
 small molecule inhibitors
 and monoclonal antibodies
 targeting CPSF73 are all potential therapeutic agents for breast cancer
siRNA small interfering RNA). siRNA is a molecule that can specifically target and
degrade the mRNA encoding CPSF73, leading to a decrease in its expression and
function
• a study published in the journal Oncotarget in 2016 showed that siRNA
targeting CPSF73 inhibited the growth of breast cancer cells and promoted
their apoptosis in vitro. Another study published in Molecular Therapy -
Nucleic Acids in 2019 demonstrated that siRNA targeting CPSF73 increased
the sensitivity of breast cancer cells to doxorubicin, a commonly used
chemotherapy drug.
• one advantage of siRNA targeting CPSF73 is its specificity, as it can
selectively target CPSF73 without affecting other cellular processes. However,
a major challenge in the clinical use of siRNA is its delivery, as it is a large,
negatively charged molecule that is rapidly degraded in the bloodstream.
Therefore, various delivery systems have been developed to enhance the
delivery of siRNA, including nanoparticles and liposomes
small molecule inhibitors
• CPSF73-IN-1, which was identified in a study published in the journal
Cell Chemical Biology in 2019. CPSF73-IN-1 is a small molecule
inhibitor that was found to selectively bind to CPSF73 and inhibit its
endonuclease activity, leading to a decrease in mRNA processing and
stability. CPSF73-IN-1 was shown to inhibit the growth of breast cancer
cells in vitro and in vivo, and was found to be well-tolerated in mice.
• Another potential CPSF73 inhibitor is a peptide known as CPSF73-i2,
which was identified in a study published in the journal Cancer Research in
2020. CPSF73-i2 is a cell-penetrating peptide that can selectively bind to
CPSF73 and inhibit its endonuclease activity, leading to reduced breast
cancer cell proliferation and increased apoptosis.
monoclonal antibodies targeting CPSF73
there are currently no monoclonal antibodies targeting CPSF73 that have
been developed or approved for clinical use.
 the development of monoclonal antibodies targeting CPSF73 poses several
challenges.
 One challenge is the identification of a suitable target epitope on CPSF73
that can be recognized by the monoclonal antibody without interfering with its
function.
 Another challenge is the potential toxicity and immunogenicity of the
monoclonal antibody, which can limit its therapeutic efficacy and safety.
In summary, while monoclonal antibodies targeting CPSF73 may hold promise
as a potential therapeutic approach for breast cancer treatment, their
development is still in the early stages, and further research is needed to evaluate
their safety and efficacy in preclinical and clinical studies.
Dosing and administration