Selective MCL-1 inhibitor ABBV-467 is efficacious in tumor models but is associated with cardiac troponin increases in patients.

BACKGROUND: MCL-1 is a prosurvival B-cell lymphoma 2 family protein that plays a critical role in tumor maintenance and survival and can act as a resistance factor to multiple anticancer therapies. Herein, we describe the generation and characterization of the highly potent and selective MCL-1 inhibitor ABBV-467 and present findings from a first-in-human trial that included patients with relapsed/refractory multiple myeloma (NCT04178902). METHODS: Binding of ABBV-467 to human MCL-1 was assessed in multiple cell lines. The ability of ABBV-467 to induce tumor growth inhibition was investigated in xenograft models of human multiple myeloma and acute myelogenous leukemia. The first-in-human study was a multicenter, open-label, dose-escalation study assessing safety, pharmacokinetics, and efficacy of ABBV-467 monotherapy. RESULTS: Here we show that administration of ABBV-467 to MCL-1-dependent tumor cell lines triggers rapid and mechanism-based apoptosis. In vivo, intermittent dosing of ABBV-467 as monotherapy or in combination with venetoclax inhibits the growth of xenografts from human hematologic cancers. Results from a clinical trial evaluating ABBV-467 in patients with multiple myeloma based on these preclinical data indicate that treatment with ABBV-467 can result in disease control (seen in 1 patient), but may also cause increases in cardiac troponin levels in the plasma in some patients (seen in 4 of 8 patients), without other corresponding cardiac findings. CONCLUSIONS: The selectivity of ABBV-467 suggests that treatment-induced troponin release is a consequence of MCL-1 inhibition and therefore may represent a class effect of MCL-1 inhibitors in human patients.

Apoptosis is a type of cell death that removes abnormal cells from the body. Cancer cells can have increased levels of MCL-1, a protein that helps cells survive and prevents apoptosis. ABBV-467 is a new drug that blocks the action of MCL-1 (an MCL-1 inhibitor) and could promote apoptosis. In animal models, ABBV-467 led to cancer cell death and delayed tumor growth. ABBV-467 was also studied in a clinical trial in 8 patients with multiple myeloma, a blood cancer. In 1 patient, ABBV-467 treatment prevented the cancer from getting any worse for 8 months. However, in 4 out of 8 patients ABBV-467 increased the levels of troponin, a protein associated with damage to the heart. This concerning side effect may impact the future development of MCL-1 inhibitors as anticancer drugs.

Cooperative phagocytosis of solid tumours by macrophages triggers durable anti-tumour responses.

In solid tumours, the abundance of macrophages is typically associated with a poor prognosis. However, macrophage clusters in tumour-cell nests have been associated with survival in some tumour types. Here, by using tumour organoids comprising macrophages and cancer cells opsonized via a monoclonal antibody, we show that highly ordered clusters of macrophages cooperatively phagocytose cancer cells to suppress tumour growth. In mice with poorly immunogenic tumours, the systemic delivery of macrophages with signal-regulatory protein alpha (SIRPα) genetically knocked out or else with blockade of the CD47-SIRPα macrophage checkpoint was combined with the monoclonal antibody and subsequently triggered the production of endogenous tumour-opsonizing immunoglobulin G, substantially increased the survival of the animals and helped confer durable protection from tumour re-challenge and metastasis. Maximizing phagocytic potency by increasing macrophage numbers, by tumour-cell opsonization and by disrupting the phagocytic checkpoint CD47-SIRPα may lead to durable anti-tumour responses in solid cancers.

Synergistic therapeutic benefit by combining the antibody drug conjugate, depatux-m with temozolomide in pre-clinical models of glioblastoma with overexpression of EGFR.

PURPOSE: Depatux-m is an antibody drug conjugate (ADC) that targets and inhibits growth of cancer cells overexpressing the epidermal growth factor receptor (EGFR) or the 2-7 deletion mutant (EGFRvIII) in tumor models in vitro and in vivo. Treatment of patients suffering from relapsed/refractory glioblastoma (GBM) with a combination of depatux-m and temozolomide (TMZ) tended to increase overall survival. As a first step to understand the nature of the interaction between the two drugs, we investigated whether the interaction was synergistic, additive or antagonistic. METHODS: The efficacy of ADCs, antibodies, TMZ and radiation was tested in xenograft models of GBM, U-87MG and U-87MG EGFRvIII. Both models express EGFR. U-87MG EGFRvIII was transduced to express EGFRvIII. Changes in tumor volume, biomarkers of cell death and apoptosis after treatment were used to measure efficacy of the various treatments. Synergism of depatux-m and TMZ was verified in three-dimensional cultures of U-87MG and U-87MG EGFRvIII by the method of Chou and Talalay. RESULTS: Combined with TMZ and radiotherapy (RT), depatux-m inhibited xenograft growth of U-87MG and U-87MG EGFRvIII more than either treatment with depatux-m or TMZ + RT. Durability of the response to depatux-m + TMZ + RT or depatux-m + TMZ was more pronounced in U-87MG EGFRvIII than in U-87MG. Efficacy of depatux-m + TMZ was synergistic in U-87MG EGFRvIII and additive in U-87MG. CONCLUSION: Adding depatux-m enhances the efficacy of standard of care therapy in preclinical models of GBM. Durability of response to depatux-m + TMZ in vivo and synergy of the drug-drug interaction correlates with the amount of antigen expressed by the tumor cells.

Nuclear rupture at sites of high curvature compromises retention of DNA repair factors.

The nucleus is physically linked to the cytoskeleton, adhesions, and extracellular matrix-all of which sustain forces, but their relationships to DNA damage are obscure. We show that nuclear rupture with cytoplasmic mislocalization of multiple DNA repair factors correlates with high nuclear curvature imposed by an external probe or by cell attachment to either aligned collagen fibers or stiff matrix. Mislocalization is greatly enhanced by lamin A depletion, requires hours for nuclear reentry, and correlates with an increase in pan-nucleoplasmic foci of the DNA damage marker γH2AX. Excess DNA damage is rescued in ruptured nuclei by cooverexpression of multiple DNA repair factors as well as by soft matrix or inhibition of actomyosin tension. Increased contractility has the opposite effect, and stiff tumors with low lamin A indeed exhibit increased nuclear curvature, more frequent nuclear rupture, and excess DNA damage. Additional stresses likely play a role, but the data suggest high curvature promotes nuclear rupture, which compromises retention of DNA repair factors and favors sustained damage.

Membrane fluctuations and acidosis regulate cooperative binding of 'marker of self' protein CD47 with the macrophage checkpoint receptor SIRPα.

Cell-cell interactions that result from membrane proteins binding weakly in trans can cause accumulations in cis that suggest cooperativity and thereby an acute sensitivity to environmental factors. The ubiquitous 'marker of self' protein CD47 binds weakly to SIRPα on macrophages, which leads to accumulation of SIRPα (also known as SHPS-1, CD172A and SIRPA) at phagocytic synapses and ultimately to inhibition of engulfment of 'self' cells - including cancer cells. We reconstituted this macrophage checkpoint with GFP-tagged CD47 on giant vesicles generated from plasma membranes and then imaged vesicles adhering to SIRPα immobilized on a surface. CD47 diffusion is impeded near the surface, and the binding-unbinding events reveal cooperative interactions as a concentration-dependent two-dimensional affinity. Membrane fluctuations out-of-plane link cooperativity to membrane flexibility with suppressed fluctuations in the vicinity of bound complexes. Slight acidity (pH 6) stiffens membranes, diminishes cooperative interactions and also reduces 'self' signaling of cancer cells in phagocytosis. Sensitivity of cell-cell interactions to microenvironmental factors - such as the acidity of tumors and other diseased or inflamed sites - can thus arise from the collective cooperative properties of flexible membranes.This article has an associated First Person interview with the first author of the paper.

Filomicelles Deliver a Chemo-Differentiation Combination of Paclitaxel and Retinoic Acid That Durably Represses Carcinomas in Liver to Prolong Survival.

Drug resistance and relapse is common in cancer treatments with chemotherapeutics, and while drug combinations with naturally occurring, differentiation-inducing retinoic acid (RA) provide remission-free cures for one type of liquid tumor, solid tumors present major problems for delivery. Here, inspired by filoviruses that can be microns in length, flexible filomicelles that self-assemble from an amphiphilic block copolymer (PEG-PCL) are shown to effectively deliver RA and paclitaxel (TAX) to several solid tumor models, particularly in the liver. These hydrophobic compounds synergistically load into the cores of the elongated micelles, and the coloaded micelles prove most effective at causing cell death, ploidy, and durable regression of tumors compared to free drugs or to separately loaded drugs. RA-TAX filomicelles also reduce mortality of human lung or liver derived cancers engrafted at liver, intraperitoneal, and subcutaneous sites in immunodeficient mice. In vitro studies show that the dual drug micelles effectively suppress proliferation while upregulating a generic differentiation marker. The results highlight the potency of dual-loaded filomicelles in killing cancer cells or else driving their differentiation away from growth.

Genome variation across cancers scales with tissue stiffness - an invasion-mutation mechanism and implications for immune cell infiltration.

Many different types of soft and solid tumors have now been sequenced, and meta-analyses suggest that genomic variation across tumors scales with the stiffness of the tumors' tissues of origin. The opinion expressed here is based on a review of current genomics data, and it considers multiple 'mechanogenomics' mechanisms to potentially explain this scaling of mutation rate with tissue stiffness. Since stiff solid tissues have higher density of fibrous collagen matrix, which should decrease tissue porosity, cancer cell proliferation could be affected and so could invasion into stiff tissues as the nucleus is squeezed sufficiently to enhance DNA damage. Diversification of a cancer genome after constricted migration is now clear. Understanding genome changes that give rise to neo-antigens is important to selection as well as to the development of immunotherapies, and we discuss engineered monocytes/macrophages as particularly relevant to understanding infiltration into solid tumors.

SIRPA-Inhibited, Marrow-Derived Macrophages Engorge, Accumulate, and Differentiate in Antibody-Targeted Regression of Solid Tumors.

Marrow-derived macrophages are highly phagocytic, but whether they can also traffic into solid tumors and engulf cancer cells is questionable, given the well-known limitations of tumor-associated macrophages (TAMs). Here, SIRPα on macrophages from mouse and human marrow was inhibited to block recognition of its ligand, the "marker of self" CD47 on all other cells. These macrophages were then systemically injected into mice with fluorescent human tumors that had been antibody targeted. Within days, the tumors regressed, and single-cell fluorescence analyses showed that the more the macrophages engulfed, the more they accumulated within regressing tumors. Human-marrow-derived macrophages engorged on the human tumors, while TAMs were minimally phagocytic, even toward CD47-knockdown tumors. Past studies had opsonized tumors in situ with antibody and/or relied on mouse TAMs but had not injected SIRPα-inhibited cells; also, unlike past injections of anti-CD47, blood parameters remained normal and safe. Consistent with tumor-selective engorge-and-accumulate processes in vivo, phagocytosis in vitro inhibited macrophage migration through micropores that mimic features of dense 3D tissue. Accumulation of SIRPα-inhibited macrophages in tumors favored tumor regression for 1-2 weeks, but donor macrophages quickly differentiated toward non-phagocytic, high-SIRPα TAMs. Analyses of macrophages on soft (like marrow) or stiff (like solid tumors) collagenous gels demonstrated a stiffness-driven, retinoic-acid-modulated upregulation of SIRPα and the mechanosensitive nuclear marker lamin-A. Mechanosensitive differentiation was similarly evident in vivo and likely limited the anti-tumor effects, as confirmed by re-initiation of tumor regression by fresh injections of SIRPα-inhibited macrophages. Macrophage motility, phagocytosis, and differentiation in vivo are thus coupled.

Engineering macrophages to eat cancer: from "marker of self" CD47 and phagocytosis to differentiation.

The ability of a macrophage to engulf and break down invading cells and other targets provides a first line of immune defense in nearly all tissues. This defining ability to "phagos" or devour can subsequently activate the entire immune system against foreign and diseased cells, and progress is now being made on a decades-old idea of directing macrophages to phagocytose specific targets, such as cancer cells. Engineered T cells provide precedence with recent clinical successes against liquid tumors, but solid tumors remain a challenge, and a handful of clinical trials seek to exploit the abundance of tumor-associated macrophages instead. Although macrophage differentiation into such phenotypes with deficiencies in phagocytic ability can raise challenges, newly recognized features of cancer cells that might be manipulated to increase the phagocytosis of those cells include ≥1 membrane protein, CD47, which broadly inhibits phagocytosis and is abundantly expressed on all healthy cells. Physical properties of the target also influence phagocytosis and again relate-via cytoskeleton forces-to differentiation pathways in solid tumors. Such pathways extend to mechanosensing by the nuclear lamina, which is known to influence signaling by soluble retinoids that can regulate the macrophage SIRPα, the receptor for CD47. Here, we highlight some of those past, present, and rapidly emerging efforts to understand and control macrophages for cancer therapy.

DNA Damage Follows Repair Factor Depletion and Portends Genome Variation in Cancer Cells after Pore Migration.

Migration through micron-size constrictions has been seen to rupture the nucleus, release nuclear-localized GFP, and cause localized accumulations of ectopic 53BP1-a DNA repair protein. Here, constricted migration of two human cancer cell types and primary mesenchymal stem cells (MSCs) increases DNA breaks throughout the nucleoplasm as assessed by endogenous damage markers and by electrophoretic "comet" measurements. Migration also causes multiple DNA repair proteins to segregate away from DNA, with cytoplasmic mis-localization sustained for many hours as is relevant to delayed repair. Partial knockdown of repair factors that also regulate chromosome copy numbers is seen to increase DNA breaks in U2OS osteosarcoma cells without affecting migration and with nucleoplasmic patterns of damage similar to constricted migration. Such depletion also causes aberrant levels of DNA. Migration-induced nuclear damage is nonetheless reversible for wild-type and sub-cloned U2OS cells, except for lasting genomic differences between stable clones as revealed by DNA arrays and sequencing. Gains and losses of hundreds of megabases in many chromosomes are typical of the changes and heterogeneity in bone cancer. Phenotypic differences that arise from constricted migration of U2OS clones are further illustrated by a clone with a highly elongated and stable MSC-like shape that depends on microtubule assembly downstream of the transcription factor GATA4. Such changes are consistent with reversion to a more stem-like state upstream of cancerous osteoblastic cells. Migration-induced genomic instability can thus associate with heritable changes.

"Marker of Self" CD47 on lentiviral vectors decreases macrophage-mediated clearance and increases delivery to SIRPA-expressing lung carcinoma tumors.

Lentiviruses infect many cell types and are now widely used for gene delivery in vitro, but in vivo uptake of these foreign vectors by macrophages is a limitation. Lentivectors are produced here from packaging cells that overexpress "Marker of Self" CD47, which inhibits macrophage uptake of cells when prophagocytic factors are also displayed. Single particle analyses show "hCD47-Lenti" display properly oriented human-CD47 for interactions with the macrophage's inhibitory receptor SIRPA. Macrophages derived from human and NOD/SCID/Il2rg-/- (NSG) mice show a SIRPA-dependent decrease in transduction, i.e., transgene expression, by hCD47-Lenti compared to control Lenti. Consistent with known "Self" signaling pathways, macrophage transduction by control Lenti is decreased by drug inhibition of Myosin-II to the same levels as hCD47-Lenti. In contrast, human lung carcinoma cells express SIRPA and use it to enhance transduction by hCD47-Lenti- as illustrated by more efficient gene deletion using CRISPR/Cas9. Intravenous injection of hCD47-Lenti into NSG mice shows hCD47 prolongs circulation, unless a blocking anti-SIRPA is preinjected. In vivo transduction of spleen and liver macrophages also decreases for hCD47-Lenti while transduction of lung carcinoma xenografts increases. hCD47 could be useful when macrophage uptake is limiting on other viral vectors that are emerging in cancer treatments (e.g., Measles glycoprotein-pseudotyped lentivectors) and also in targeting various SIRPA-expressing tumors such as glioblastomas.

Macrophage engulfment of a cell or nanoparticle is regulated by unavoidable opsonization, a species-specific 'Marker of Self' CD47, and target physical properties.

Professional phagocytes of the mononuclear phagocyte system (MPS), especially ubiquitous macrophages, are commonly thought to engulf or not a target based strictly on 'eat me' molecules such as Antibodies. The target might be a viable 'self' cell or a drug-delivering nanoparticle, or it might be a cancer cell or a microbe. 'Marker of Self' CD47 signals into a macrophage to inhibit the acto-myosin cytoskeleton that makes engulfment efficient. In adhesion of any cell, the same machinery is generally activated by rigidity of target surfaces, and recent results confirm phagocytosis is likewise driven by the rigidity typical of microbes and many synthetics. Basic insights are already being applied in order to make macrophages eat cancer or to delay nanoparticle clearance for better drug delivery and imaging.

One-bead, one-compound peptide library sequencing via high-pressure ammonia cleavage coupled to nanomanipulation/nanoelectrospray ionization mass spectrometry.

Biological screening of one-bead, one-compound (OBOC) combinatorial peptide libraries is routinely carried out with the peptide remaining bound to the resin bead during screening. After a hit is identified, the bead is isolated, the peptide is cleaved from the bead, and its sequence is determined. We have developed a new technique for cleavage of peptides from resin beads whereby exposure of a 4-hydroxymethyl benzoic acid (HMBA)-linked peptide to high-pressure ammonia gas led to efficient cleavage in as little as 5min. Here we also report a new method of extracting peptide from individual library beads for its introduction into a mass spectrometer that uses nanomanipulation combined with nanoelectrospray ionization mass spectrometry (NSI MS). Single beads analyzed by nanomanipulation/NSI MS were found to give identical MS results to those of bulk samples. Detection of 18 unique cleaved peptides 1 to 8 amino acids in length, and sequencing of 14 different peptide sequences 4 to 8 amino acids in length, was demonstrated on a combination of bulk samples and ones from individual beads of an OBOC library. The method was highly reproducible, with 100% of attempts to extract peptide resulting in high-quality MS data. This new collection of techniques allows rapid, reliable, environmentally responsible sequencing of hit beads from combinatorial peptide libraries.