BCR-ABL residues interacting with ponatinib are critical to preserve the tumorigenic potential of the oncoprotein

Patients with chronic myeloid leukemia in whom tyrosine kinase inhibitors (TKIs) fail often present mutations in the BCR-ABL catalytic domain. We noticed a lack of substitutions involving 4 amino acids (E286, M318, I360, and D381) that form hydrogen bonds with ponatinib. We therefore introduced mutations in each of these residues, either preserving or altering their physicochemical properties. We found that E286, M318, I360, and D381 are dispensable for ABL and BCR-ABL protein stability but are critical for preserving catalytic activity. Indeed, only a "conservative" I360T substitution retained kinase proficiency and transforming potential. Molecular dynamics simulations of BCR-ABLI360T revealed differences in both helix αC dynamics and protein-correlated motions, consistent with a modified ATP-binding pocket. Nevertheless, this mutant remained sensitive to ponatinib, imatinib, and dasatinib. These results suggest that changes in the 4 BCR-ABL residues described here would be selected against by a lack of kinase activity or by maintained responsiveness to TKIs. Notably, amino acids equivalent to those identified in BCR-ABL are conserved in 51% of human tyrosine kinases. Hence, these residues may represent an appealing target for the design of pharmacological compounds that would inhibit additional oncogenic tyrosine kinases while avoiding the emergence of resistance due to point mutations.This work was supported by an investigator grant to P.V. from Associazione Italiana per la Ricerca sul Cancro (AIRC) and by funding from the Biotechnology and Biological Sciences Research Council (BB/I023291/1 and BB/H018409/1 to AP and FF). P.B. is the recipient of an AIRC - Marie Curie fellowship

Chk1 Inhibition Restores Inotuzumab Ozogamicin Citotoxicity in CD22-Positive Cells Expressing Mutant p53

Inotuzumab ozogamicin (IO) is an anti-CD22 calicheamicin immunoconjugate that has been recently approved for the treatment of relapsed or refractory B-Acute Lymphoblastic Leukemia (r/r B-ALL). We employed both immortalized and primary cells derived from CD22-positive lymphoproliferative disorders to investigate the signaling pathways contributing to IO sensitivity or resistance. We found that the drug reduced the proliferation rate of CD22-positive cell lines expressing wild-type p53, but was remarkably less effective on cells exhibiting mutant p53. In addition, CD22-positive cells surviving IO were mostly blocked in the G2/M phase of the cell cycle because of Chk1 activation that, in the presence of a wild-type p53 background, led to p21 induction. When we combined IO with the Chk1 inhibitor UCN-01, we successfully abrogated IO-induced G2/M arrest regardless of the underlying p53 status, indicating that the DNA damage response triggered by IO is also modulated by p53-independent mechanisms. To establish a predictive value for p53 in determining IO responsiveness, we expressed mutant p53 in cell lines displaying the wild-type gene and observed an increase in IO IC50 values. Likewise, overexpression of an inducible wild-type p53 in cells natively presenting a mutant protein decreased their IC50 for IO. These results were also confirmed in primary CD22-positive cells derived from B-ALL patients at diagnosis and from patients with r/r B-ALL. Furthermore, co-treatment with IO and UCN-01 significantly increased cell death in primary cells expressing mutant p53. In summary, our findings suggest that p53 status may represent a biomarker predictive of IO efficacy in patients diagnosed with CD22-positive malignancies

New Insights in Thyroid Cancer and p53 Family Proteins

Thyroid cancers are common endocrine malignancies that comprise tumors with different clinical and histological features. Indeed, papillary and follicular thyroid cancers are slow-growing, well-differentiated tumors, whereas anaplastic thyroid cancers are undifferentiated neoplasias that behave much more aggressively. Well-differentiated thyroid carcinomas are efficiently cured by surgery and radioiodine, unlike undifferentiated tumors that fail to uptake radioactive iodine and are usually resistant to chemotherapy. Therefore, novel and more effective therapies for these aggressive neoplasias are urgently needed. Whereas most genetic events underlying the pathogenesis of well-differentiated thyroid cancers have been identified, the molecular mechanisms that generate undifferentiated thyroid carcinomas are still unclear. To date, one of the best-characterized genetic alterations leading to the development of poorly differentiated thyroid tumors is the loss of the p53 tumor suppressor gene. In addition, the existence of a complex network among p53 family members (p63 and p73) and their interactions with other factors that promote thyroid cancer progression has been well documented. In this review, we provide an update on the current knowledge of the role of p53 family proteins in thyroid cancer and their possible use as a therapeutic target for the treatment of the most aggressive variants of this disease

Additional Genetic Alterations and Clonal Evolution of MPNs with Double Mutations on the <i>MPL</i> Gene: Two Case Reports

Essential thrombocythemia (ET) and primary myelofibrosis (PMF) are two of the main BCR-ABL1-negative chronic myeloproliferative neoplasms (MPNs) characterized by abnormal megakaryocytic proliferation. Janus kinase 2 (JAK2) mutations are detected in 50–60% of ET and PMF, while myeloproliferative leukemia (MPL) virus oncogene mutations are present in 3–5% of cases. While Sanger sequencing is a valuable diagnostic tool to discriminate the most common MPN mutations, next-generation sequencing (NGS) is a more sensitive technology that also identifies concurrent genetic alterations. In this report, we describe two MPN patients with simultaneous double MPL mutations: a woman with ET presenting both MPLV501A-W515R and JAK2V617F mutations and a man with PMF displaying an uncommon double MPLV501A-W515L. Using colony-forming assays and NGS analyses, we define the origin and mutational landscape of these two unusual malignancies and uncover further gene alterations that may contribute to the pathogenesis of ET and PMF

Orbital metastasis from thyroid cancer: a case report and review of the literature.

BACKGROUND: Differentiated thyroid cancer (DTC) is generally associated with an excellent prognosis. However up to 20% of DTC patients have disease events during subsequent follow-up; rarely patients present an aggressive disease with distant metastases (DM), mainly in the lung and bone. Metastases at unusual sites may also occur, generally in patients with disseminated disease. Orbital localization is rare and only few cases have been described so far. CASE DESCRIPTION: A 36 years-old man, treated with chemo and radiotherapy during childhood for non-Hodgkin lymphoma, was referred for suspicious lymph node (LN) and multiple lung metastases. Total thyroidectomy and latero-cervical (LC) lymphadenectomy were performed: papillary thyroid cancer (PTC), 25 mm, 11/17 LN metastases; pT2N1bM1. Post-treatment total body scan with I-131 showed LN and lung uptake. Eighteen months from diagnosis he presented progressive diplopia, proptosis and right exophthalmos due to an 18 mm orbital metastasis. Hence, due to I-131 refractoriness for structural disease progression despite I-131 therapy, he started therapy with Lenvatinib for 6 months, with initial partial response followed by disease progression, and then with Cabozantinib, which he stopped after 6 months for adverse events and disease progression after therapy reduction. Currently, the patient is receiving Lenvatinib, rechallenge therapy, with disease stabilization and biochemical response. Molecular analysis, performed on both primary and relapsed tumor didn't show any significant pathogenic alteration. CONCLUSIONS: This case of DTC with an unusual metastasis in the orbit, may suggest that patient's exposure to chemo- and radiotherapy during pediatric age might have played a role in the subsequent development of this unusually aggressive tumor, reinforcing the recommendation of long-term and intensive follow-up of these patients

Combined Inhibition of Bcl2 and Bcr-Abl1 Exercises Anti-Leukemia Activity but Does Not Eradicate the Primitive Leukemic Cells

Background: The management of Philadelphia Chromosome-positive (Ph+) hematological malignancies is strictly correlated to the use of BCR-ABL1 tyrosine kinase inhibitors (TKIs). However, these drugs do not induce leukemic stem cells death and their persistence may generate a disease relapse. Published reports indicated that Venetoclax, a selective BCL2 inhibitor, could be effective in Ph+ diseases, as BCL2 anti-apoptotic activity is modulated by BCR-ABL1 kinase. We, therefore, investigated if BCL2 inhibition, alone or combined with Nilotinib, a BCR-ABL1 inhibitor, affects the primitive and committed Ph+ cells survival. Methods: We used Ph+ cells isolated from leukemic patients at diagnosis. To estimate the therapeutic efficacy of BCL2 and BCR-ABL1 inhibition we employed long-term culture, proliferation and apoptosis assay. Immunoblot was used to evaluate the ability of treatment to interfere with the down-stream targets of BCR-ABL1. Results: Blocking BCL2, we observed reduced proliferation and clonogenic potential of CML CD34-positive cells and this cytotoxicity was improved by combination with BCR-ABL1 inhibitor. However, BCL2 inhibition, alone or in combination regiment with BCR-ABL1 inhibitor, did not reduce the self-renewal of primitive leukemic cells, while strongly induced cell death on primary Ph+ Acute Lymphoblastic Leukemia (ALL). Conclusion: Our results suggest that primitive CML leukemic cells are not dependent on BCL2 for their persistence and support that committed CML and Ph + ALL cells are dependent by BCL2 and BCR-ABL1 cooperation for their survival. The antileukemic activity of BCL2 and BCR-ABL1 dual targeting may be a useful therapeutic strategy for Ph+ ALL patients

Non ABL-directed inhibitors as alternative treatment strategies for chronic myeloid leukemia

Abstract The introduction of ABL Tyrosine Kinase Inhibitors (TKIs) has significantly improved the outcome of Chronic Myeloid Leukemia (CML) patients that, in large part, achieve satisfactory hematological, cytogenetic and molecular remissions. However, approximately 15–20% fail to obtain optimal responses according to the current European Leukemia Network recommendation because of drug intolerance or resistance. Moreover, a plethora of evidence suggests that Leukemic Stem Cells (LSCs) show BCR-ABL1-independent survival. Hence, they are unresponsive to TKIs, leading to disease relapse if pharmacological treatment is discontinued. All together, these biological events generate a subpopulation of CML patients in need of alternative therapeutic strategies to overcome TKI resistance or to eradicate LSCs in order to allow cure of the disease. In this review we update the role of “non ABL-directed inhibitors” targeting signaling pathways downstream of the BCR-ABL1 oncoprotein and describe immunological approaches activating specific T cell responses against CML cells

A Case of Non-Irradiated Balloon Cell Melanoma of the Choroid: Expanding the Morphological Spectrum of Primary Uveal Melanomas

Uveal melanoma (UM) is the most common primary intraocular tumor in adults and usually has a very poor prognosis. Histologically, UMs have been classified in epithelioid cell type, spindle cell type, and mixed cell type. Balloon cells are large pale cells that contain small, hyperchromatic, central nuclei with vesiculated, clear, and lipid-rich cytoplasm. A balloon cell morphology is infrequently observed in naevi and even less frequently in malignant melanomas of the skin, conjunctiva, ciliary body and choroid. In this regard, UMs that exhibit balloon cell features are generally those previously treated with proton beam irradiation and then enucleated, rather than those that directly underwent primary surgery. To the best of our knowledge, very few cases of primary UM showing extensive balloon cell morphology have been reported in scientific literature to date. We herein present an unusual case of primary UM with diffuse balloon cell changes in a 69-year-old woman