Preclinical analysis of MAD28 in inflammatory breast cancer

Institution: University of California, San Diego
Investigator(s): Emmanuel Theodorakis, Ph.D. -
Award Cycle: 2016 (Cycle 22) Grant #: 22IB-0024 Award: $210,715
Award Type: IDEA
Research Priorities
Detection, Prognosis and Treatment>Innovative Treatment Modalities: search for a cure

Initial Award Abstract (2016)

Introduction: According to the American Cancer Society approximately 200,000 women are diagnosed every year with breast cancer and about 40,000 of them will die from this disease. Among the new breast cancer cases, about 3-10% belong to a highly aggressive and rapidly metastasizing subtype, referred to as inflammatory breast cancer (IBC), that is characterized by a younger age of onset and a higher incidence in African-American women. At present, there is no effective treatment of IBC and, despite recent conventional, multimodality therapies, its mortality rate remains high. In fact, IBC is the most lethal form of breast cancer with a 5-year survival rate of only 40% (for comparison the survival rate of non-IBC patients is more than 85%). The overarching goal of this research is to evaluate the preclinical pharmacology of a recently identified small molecule as a potential IBC therapeutic strategy thereby providing a new and highly promising approach in the management and treatment of this very lethal disease.

Significance: The importance of studying IBC is underlined by the finding that: (a) IBC is the most lethal form of breast cancer with a 5-year survival rate of only 40%; (b) non-IBC primary lesions often recur with the IBC signature phenotype; and (c) locally advanced non-IBC successfully treated with neoadjuvant chemotherapy often show residual emboli exclusively in the lymphovasculature. Therefore, therapies that address IBC will potentially be applicable to all breast cancers that do not remain organ-confined. Developing novel, more effective and less toxic therapeutics towards IBC provides a promising new approach in the management and treatment of this very lethal disease i.e. the ‘worst case scenario’ of breast cancers, and most significantly, will potentially provide insight and novel therapeutics for all breast cancers that do not remain organ-confined.

Hypothesis: Mitochondria are cellular organelles that regulate key cellular processes that differ at their functional and structural levels between normal and cancer cell. Significantly, IBC displays structurally different mitochondria (i.e. hyperfused or increased mitochondrial network) suggesting that this organelle potentially contributes in the IBC aggressive nature. Taking advantage of the high functional activity of IBC mitochondria, we propose to investigate the use of a recently discovered mitochondriotoxic molecule (i.e. MAD28) for the treatment of this disease. Our working hypothesis is that mitochondriotoxic molecules such as MAD28 represent a novel therapeutic approach against IBC.

Methodology: Using our preclinical IBC model, MARY-X, that precisely captures the hallmark feature of IBC, where the cancer cells grow exclusively in the mouse lymphatic and blood vessels, we performed an in vitro screen of a library of synthetic molecules to ascertain new therapeutic strategies in the treatment of IBC. Screening led to the discovery of MAD28, a new molecule that induces rapid dissolution of the characteristic intact IBC spheroids/emboli leading to cell death. Importantly, such an effect has not been observed with other commonly used anticancer drugs attesting to the novelty and significance of the proposed research.

Innovative Elements: Inflammatory breast cancer (IBC) represents the most lethal and the least understood subtype of breast cancer. Its high lethality originates from: (a) the absence of a palpable tumor mass that hinders early diagnosis; and (b) a highly metastatic nature. It should be noted that IBC patients have benefited little from advances made in therapeutic strategies for the treatment of breast cancer, including chemotherapy, surgery, radiotherapy and combinations thereof. Unfortunately all current chemotherapeutic approaches against IBC are either destructive to both cancer and noncancerous cells (e.g. anthracyclines) or not effective in clinical trials (e.g. farnesyltransferase inhibitors). Thus, new mechanism-based chemotherapeutic agents are needed. The proposed approach combines the innovation of MARY-X, the only IBC preclinical model that precisely captures the signature phenotype of the disease, with a state-of-the-art medicinal chemistry program to develop clinical candidates against IBC. Preliminary results in support of our approach led to the identification of a new small molecule, known as MAD28 that exhibits potent and selective cytotoxicity against IBC in vitro and is now at the forefront of preclinical pharmacological evaluation, as proposed herein.

Final Report (2019)

Inflammatory breast cancer (IBC) represents the most lethal and least understood form of primary breast cancer for which, at-present, there is no effective chemotherapeutic treatment. The overarching goal of this research is to chemically and pharmacologically optimize a small molecule, referred to as MAD28, as a novel and potent lead against IBC, thereby providing a new and highly promising chemotherapeutic lead to treat this very lethal disease. Along these lines, the milestones of this proposal are to (a) create a focused library of MAD28 analogs to enhance function and potency in spheroidsMARY-X; (b) evaluate the stability and perform plasma pharmacokinetic profiling and maximum tolerated dose of MAD28 and lead analogs; and (c) to evaluate the efficacy of selected MAD analogs in the IBC preclinical model, MARY-X.

In an effort to improve function and potency of MAD28, last year we designed and synthesized 10 analogues that have various functionalities at the periphery of the central pharmacophore. Our design criteria included conjugation of the pharmacophore with side chains that enhance solubility and tissue permeability. These analogues were screened in an assay, referred to as spheroidsMARY-X, that represents the most relevant in vitro screening platform for the discovery of new anticancer drugs specific to IBC. This screening led to the identification of CR136 and MAD28 as the most potent leads, thus completing a significant milestone of this proposal. Since both compounds were synthesized and screened as racemates, in subsequent studies we resolved the two enantiomers of MAD28. We have completed the aims of this proposal.

Major accomplishments of this research include: (a) the construction of a focused chemical library of MAD28 and its screening in the spheroidsMARY-X assay; (b) the development of a strategy for the racemic resolution of MAD28; and (c) the finding that both MAD28 enantiomers have similar biological profile and cytotoxicity. The first accomplishment allowed a methodical structure-activity relationship study leading to the identification of CR136 as a potent lead. The latter accomplishment is significant since on several occasions the enantiomers have different targets and pharmacological profile, resulting in unwanted side effects when administered together. The third accomplishment is significant because it facilitates the synthesis, since we no longer need to resolve the two enantiomers in order to achieve potency and efficacy. Overall, our results show that MAD28 is selectively cytotoxic against breast cancer cells with a triple negative phenotype. As such, it represents an excellent lead for drug development against metastatic and aggressive breast cancers. We plan to submit a proposal in the near future to further explore this selective cytotoxicity and pharmacology.